Antibody drug conjugates

ABSTRACT

The present invention relates to anti-FGFR2 and FGFR4 antibodies, antibody fragments, antibody drug conjugates, and their uses for the treatment of cancer.

This application is a divisional of U.S. patent application Ser. No.14/203,997, filed Mar. 11, 2014, which claims priority to U.S.Provisional Application No. 61/780,299, filed Mar. 13, 2013, thecontents of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 30, 2014, isnamed PAT055642-US-NP_SL.txt and is 169,317 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to anti-FGFR2 and FGFR4antibodies, antibody fragments, antibody drug conjugates, and their usesfor the treatment of cancer.

BACKGROUND OF THE INVENTION Fibroblast Growth Factor Receptors

Fibroblast growth factors (FGFs) that signal through FGF receptors(FGFRs) regulate fundamental developmental pathways and are expressed ina wide variety of tissues. FGFs stimulate proliferation, cell migration,differentiation, and play a major role in skeletal and limb development,wound healing, tissue repair, hematopoiesis, angiogenesis, andtumorigenesis (see, e.g., Turner and Crose, Nature Reviews Cancer 10:116-129 (2010)).

The mammalian FGF family comprises many ligands, which exert theiractions through four highly conserved transmembrane tyrosine kinasereceptors, FGFR1, FGFR2, FGFR3, and FGFR4. A typical FGFR has a signalpeptide that is cleaved off, three immunoglobulin (Ig)-like domains (Igdomain I, II and III), an acidic box, a transmembrane domain, and asplit tyrosine kinase domain (see, e.g., Ullrich and Schlessinger, Cell61: 203 (1990); Johnson and Williams, Adv. Cancer Res. 60: 1-41 (1992)).

Additionally, alternative splicing of the transcribed receptor genesresults in a variety of receptor isoforms, including soluble, secretedFGFRs, FGFRs with truncated COOH-terminal domain, FGFRs with either twoor three Ig-like domains, as well as FGFR isoforms arising viaalternative splicing of the third Ig-like domain occurs only for FGFR1,FGFR2, and FGFR3 and specifies the second half of the third Ig-likedomain, resulting in either the IIIb or the IIIc isoform of thereceptor. The 2^(nd) and the 3^(rd) Ig-like domains of the receptors arenecessary and sufficient for ligand binding, whereas the first Ig-likedomain is thought to play a role in receptor autoinhibition. Thus, thedifferent receptors and their isoforms display different ligand-bindingspecificities (see, e.g., Haugsten et al., Mol. Cancer Res. 8:1439-1452(2010)).

The FGFs can also bind to heparin sulfate proteoglycans (HSPG), besidesbinding to distinct FGFRs and their splice variants. Thereby a dimericFGF-FGFR-HSPG ternary complex forms on the cell surface. The ternarycomplex is stabilized by multiple interactions between the differentcomponents in the complex. Two FGF-binding sites, a heparin-bindingsite, and a receptor-receptor interaction site have been identifiedwithin the Ig-like domains II and III of the receptor. (Haugsten et al.,2010).

Binding of FGFs to FGFRs induces receptor dimerization, which enablestransphosphorylation of a tyrosine in the activation loop of the kinasedomain. The active FGFRs have been shown to phosphorylate multipleintracellular proteins such as FRS2, and PLCy (Eswarakumar et al.,Cytokine Growth Factor Rev 16:139-149 (2005)). FGFR signaling producesdistinct biological responses in different cell types, ranging fromstimulation of cell proliferation and survival to growth arrest,migration, and differentiation.

FGFRs and Cancer

FGF signaling mediates a powerful combination of effects:self-sufficiency in growth/survival, neoangiogenesis and tumor cellmigration. Consequently, FGF signaling has the potential of beingstrongly oncogenic once the tight regulation exerted on itsphysiological functions is lost (see, e.g., Heinzle et al., Expert Opin.Ther. Targets 15(7):829-846 (2011)).

Gene amplification and/or overexpression of FGFR1, FGFR2 and FGFR4 hasbeen implicated in breast cancer (Penault-Llorca et al., Int J Cancer1995; Theillet et al., Genes Chrom. Cancer 1993; Adnane et al., Oncogene1991; Jaakola et al., Int J Cancer 1993; Yamada et al., Neuro Res 2002).Overexpression of FGFR1 and FGFR4 is also associated with pancreaticadenocarcinomas and astrocytomas (Kobrin et al., Cancer Research 1993;Yamanaka et al., Cancer Research 1993; Shah et al., Oncogene 2002;Yamaguchi et al., PNAS 1994; Yamada et al., Neuro Res 2002). Prostatecancer has also been related to FGFR1 overexpression (Giri et al., ClinCancer Res 1999).

In general FGFR2 and FGFR1 are more commonly deregulated by geneamplification. In gastric cancer, an amplified FGFR2 gene is associatedwith poor prognosis (Kunii et al., Cancer Res. 68:2340-2348 (2008)). AFGFR2-IIIb to IIIc switch can also be a sign of tumor progression,epithelial-mesenchymal transition and high invasiveness in bladder andprostate cancers. Switching in this case substitutes a IIIb receptorvariant that exerts anti-tumorigenic activity with a protumorigenic IIIcreceptor (see, e.g., Heinzle et al., 2011). So far FGFR2 mutations havebeen implicated in skin, endometrial, ovary, and lung cancer. FGFR4mutations have been implicated in rhabdomyosarcoma, lung, and breastcancer (see, e.g., Heinzle et al., 2011).

Antibody Drug Conjugates

Antibody drug conjugates (“ADCs”) have been used for the local deliveryof cytotoxic agents in the treatment of cancer (see e.g., Lambert, Curr.Opinion In Pharmacology 5:543-549, 2005). ADCs allow targeted deliveryof the drug moiety where maximum efficacy with minimal toxicity may beachieved. As more ADCs show promising clinical results, there is anincreased need to develop new therapeutics for cancer therapy.

SUMMARY OF THE INVENTION

The present invention provides antibody drug conjugates of the formulaAb-(L-(D)_(m))_(n), or a pharmaceutically acceptable salt thereof,wherein Ab is an antibody or antigen binding fragment thereof thatspecifically binds to both human FGFR2 and FGFR4; L is a linker; D is adrug moiety; m is an integer from 1 to 8; and n is an integer from 1 to10. In some embodiments, m is 1. In some embodiments, n is 3 or 4. In aspecific embodiment, m is 1 and n is 3 or 4.

The present invention provides antibodies or antigen binding fragmentsthereof that specifically binds to human FGFR2 and human FGFR4. In someembodiments, the antibodies or antigen binding fragments describedherein specifically bind to human FGFR4 and all isoforms of human FGFR2.In some embodiment, the antibodies or antigen binding fragmentsdescribed herein specifically bind to an epitope on human FGFR2comprising amino acid residues 176 (Lys) and 210 (Arg) of SEQ ID NO:137. In some embodiments, the antibodies or antigen binding fragmentsdescribed herein recognize amino acid residues 173 (Asn), 174 (Thr), 175(Val), 176 (Lys), 178 (Arg), 208 (Lys), 209 (Val), 210 (Arg), 212 (Gln),213 (His), 217 (Ile), and 219 (Glu) of SEQ ID NO: 137. The presentinvention further provides antibody drug conjugates comprising theantibodies or antigen binding fragments described herein.

In some embodiments, the antibodies or antigen binding fragmentsdescribed herein specifically bind to an epitope of human FGFR2comprising or consisting SEQ ID NO: 136 or SEQ ID NO: 141. The presentinvention further provides antibody drug conjugates comprising theantibodies or antigen binding fragments described herein.

The antibody, antigen binding fragments, and the antibody drugconjugates of the present invention also specifically bind to humanFGFR4. In some embodiments, they specifically bind to D1 and D2 domainsof human FGFR4. In some embodiments, they specifically bind to D1 or D2domain of human FGFR4.

In some embodiments, the present invention provides antibodies describedin Table 1, antigen binding fragments thereof, and antibody drugconjugates comprising such antibodies or antigen binding fragments. Insome embodiments, the present invention provides antibodies and antigenbinding fragments comprising a heavy chain variable region thatcomprises: (a) a VH CDR1 of SEQ ID NO: 1, (b) a VH CDR2 of SEQ ID NO: 2,and (c) a VH CDR3 of SEQ ID NO: 3, wherein the CDR is defined inaccordance with the Kabat definition. In some embodiments, theantibodies or antigen binding fragments of the invention comprise aheavy chain variable region that comprises: (a) a VH CDR1 of SEQ ID NO:1, (b) a VH CDR2 of SEQ ID NO: 2, and (c) a VH CDR3 of SEQ ID NO: 3; anda light chain variable region that comprises: (a) a VL CDR1 of SEQ IDNO: 11, (b) a VL CDR2 of SEQ ID NO: 12, and (c) a VL CDR3 of SEQ ID NO:13, wherein the CDR is defined in accordance with the Kabat definition.The present invention further provides antibody drug conjugates thatcomprise such antibodies or antigen binding fragments.

In some embodiments, the antibodies or antigen binding fragmentsdescribed herein comprise a VH region of SEQ ID NO: 7 and a VL region ofSEQ ID NO: 17. In some embodiments, the antibodies described hereinconsist of a heavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO:19. The present invention also provides antibody drug conjugatescomprising such antibodies or antigen binding fragments thereof.

In some embodiments, the antibodies or antigen binding fragmentsdescribed herein are antibodies or antigen fragments that cross competewith an antibody consists of a heavy chain of SEQ ID NO: 9 and a lightchain of SEQ ID NO: 19 for binding to human FGFR2 and FGFR4. The presentinvention also provides antibody drug conjugates comprising suchantibodies or antigen binding fragments.

In some embodiments, the antibodies or antigen binding fragmentsdescribed herein have enhanced ADCC activity as compared to an antibodyconsisting of a heavy chain of SEQ ID NO: 9 and a light chain of SEQ IDNO: 19. In some embodiments, the antibodies or antigen binding fragmentsdescribed herein do not have enhanced ADCC activity as compared to anantibody consisting of a heavy chain of SEQ ID NO: 9 and a light chainof SEQ ID NO: 19. The present invention also provides antibody drugconjugates comprising such antibodies or antigen binding fragments.

In some embodiments, the antibodies described herein are human orhumanized antibodies. In some embodiments, the antibodies describedherein are monoclonal antibodies. In some embodiments, the antibodiesdescribed herein are monoclonal human or humanized antibodies.

In some embodiments, the present invention provides antibody drugconjugates comprising an antibody or antigen binding fragment describedherein, and a drug moiety, wherein the drug moiety is linked to theantibody or antigen binding fragment via a linke, and wherein saidlinker is selected from the group consisting of a cleavable linker, anon-cleavable linker, a hydrophilic linker, a procharged linker and adicarboxylic acid based linker. In some embodiments, the linker isderived from a cross-linking reagent selected from the group consistingof: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1). In a specific embodiment, the linker is derived from thecross-linking reagent N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).

In some embodiments, the present invention provides antibody drugconjugates comprising an antibody or antigen binding fragment describedherein, and a drug moiety, wherein said drug moiety is selected from agroup consisting of: a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, anmTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, anauristatin, a dolastatin, a maytansinoid, a MetAP (methionineaminopeptidase), an inhibitor of nuclear export of proteins CRM1, aDPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryltransfer reactions in mitochondria, a protein synthesis inhibitor, akinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesininhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylatingagent, a DNA intercalator, a DNA minor groove binder and a DHFRinhibitor. In a specific embodiment, the drug moiety of an antibody drugconjugate of the invention is maytansinoid. In another specificembodiment, the drug moiety of an antibody drug conjugate of theinvention is N(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine(DM1) or N(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine(DM4).

In one embodiment, the present invention provides antibody drugconjugates having the following formula:

wherein Ab is an antibody or antigen binding fragment thereof comprisinga heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2,a heavy chain CDR3 of SEQ ID NO: 3, and a light chain CDR1 of SEQ ID NO:11, a light chain CDR2 of SEQ ID NO: 12, a light chain CDR3 of SEQ IDNO: 13, wherein the CDR is defined in accordance with the Kabatdefinition; and n is 1 to 10; or a pharmaceutically acceptable saltthereof. In a specific embodiment, n is 3 or 4. In one embodiment, Ab isan antibody comprising a VH region of SEQ ID NO: 7 and a VL region ofSEQ ID NO: 17. In another embodiment, Ab is an antibody consist of aheavy chain of SEQ ID NO: 9 and a light chain of SEQ ID NO: 19.

The present invention also provides pharmaceutical compositionscomprising the antibody drug conjugate described herein and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical compositions of the invention is prepared as alyophilisate. In some embodiments, the lyophilisate comprises anantibody drug conjugate described herein, sodium succinate, andpolysorbate 20.

The present invention provides methods of treating an FGFR2 positive orFGFR4 positive cancer in a patient in need thereof, comprisingadministering to said patient an antibody drug conjugate or apharmaceutical composition described herein. In some embodiments, thecancer is selected from the group consisting of gastric cancer, breastcancer, alveolar rhabdomyosarcoma, liver cancer, adrenal cancer, lungcancer, colon cancer and endometrial cancer. In some embodiments, thetreatment methods described herein further comprise administering tosaid patient a tyrosine kinase inhibitor, an IAP inhibitor, a Bcl2inhibitor, an MCL1 inhibitor, or another FGFR2 inhibitor. In a specificembodiment, the treatment method comprising administering to a patientin need thereof an antibody drug conjugate described herein incombination with3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea.

The present invention further provides antibody drug conjugatesdescribed herein for use as a medicament. The present invention providesantibody drug conjugates or pharmaceutical compositions for use in thetreatment of an FGFR2 positive or FGFR4 positive cancer.

In some embodiments, the antibodies or antigen binding fragmentsdescribed herein are a single chain antibody (scFv).

The present invention also provides nucleic acids that encodes theantibodies or antigen binding fragments described herein. In someembodiments, the present invention provides nucleic acids that encodesthe antibodies or antigen binding fragments as those described inTable 1. The present invention further provides vectors comprising thenucleic acid described herein, and host cells comprising such vectors.

The present invention provides processes for producing an antibody orantigen binding fragment comprising cultivating the host cells describedherein and recovering the antibody from the culture.

In one embodiment, the present invention provides a process forproducing an anti-FGFR2 and FGFR4 antibody drug conjugate comprising:(a) chemically linking SMCC to a drug moiety DM-1; (b) conjugating saidlinker-drug to the antibody recovered from the cell culture; and (c)purifying the antibody drug conjugate. In some embodiments, the antibodydrug conjugates made according the process have an average DAR, measuredwith a UV spectrophotometer, about 3.5.

The present invention also provides diagnostic reagents comprising anantibody or antigen binding fragment described herein, or the antibodydrug conjugate described herein which is labeled. In some embodiments,the label is selected from the group consisting of a radiolabel, afluorophore, a chromophore, an imaging agent, and a metal ion.

DEFINITIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

The term “alkyl” refers to a monovalent saturated hydrocarbon chainhaving the specified number of carbon atoms. For example, C₁₋₆ alkylrefers to an alkyl group having from 1 to 6 carbon atoms. Alkyl groupsmay be straight or branched. Representative branched alkyl groups haveone, two, or three branches. Examples of alkyl groups include, but arenot limited to, methyl, ethyl, propyl (n-propyl and isopropyl), butyl(n-butyl, isobutyl, sec-butyl, and t-butyl), pentyl (n-pentyl,isopentyl, and neopentyl), and hexyl.

The term “antibody” as used herein refers to a polypeptide of theimmunoglobulin family that is capable of binding a corresponding antigennon-covalently, reversibly, and in a specific manner. For example, anaturally occurring IgG antibody is a tetramer comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as VH) and a heavy chain constant region. The heavychain constant region is comprised of three domains, CH1, CH2 and CH3.Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy andlight chains contain a binding domain that interacts with an antigen.The constant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antibody” includes, but is not limited to, monoclonalantibodies, human antibodies, humanized antibodies, chimeric antibodies,and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention). The antibodies can be of anyisotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

“Complementarity-determining domains” or “complementary-determiningregions (“CDRs”) interchangeably refer to the hypervariable regions ofVL and VH. The CDRs are the target protein-binding site of the antibodychains that harbors specificity for such target protein. There are threeCDRs (CDR1-3, numbered sequentially from the N-terminus) in each humanVL or VH, constituting about 15-20% of the variable domains. The CDRsare structurally complementary to the epitope of the target protein andare thus directly responsible for the binding specificity. The remainingstretches of the VL or VH, the so-called framework regions, exhibit lessvariation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4.W.H. Freeman & Co., New York, 2000).

The positions of the CDRs and framework regions can be determined usingvarious well known definitions in the art, e.g., Kabat, Chothia,international ImMunoGeneTics database (IMGT) (on the worldwide web atimgt.cines.fr/), and AbM (see, e.g., Johnson et al., Nucleic Acids Res.,29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987);Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol.Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol.,273:927-748 (1997)). Definitions of antigen combining sites are alsodescribed in the following: Ruiz et al., Nucleic Acids Res., 28:219-221(2000); and Lefranc, M. P., Nucleic Acids Res., 29:207-209 (2001);MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al.,Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., MethodsEnzymol., 203:121-153 (1991); and Rees et al., In Sternberg M. J. E.(ed.), Protein Structure Prediction, Oxford University Press, Oxford,141-172 (1996).

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention, the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminus is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminal domains of the heavy and light chain,respectively.

The term “antigen binding fragment”, as used herein, refers to one ormore portions of an antibody that retain the ability to specificallyinteract with (e.g., by binding, steric hindrance,stabilizing/destabilizing, spatial distribution) an epitope of anantigen. Examples of binding fragments include, but are not limited to,single-chain Fvs (scFv), camelid antibodies, disulfide-linked Fvs(sdFv), Fab fragments, F(ab′) fragments, a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; a Fd fragment consisting of the VH and CH1domains; a Fv fragment consisting of the VL and VH domains of a singlearm of an antibody; a dAb fragment (Ward et al., Nature 341:544-546,1989), which consists of a VH domain; and an isolated complementaritydetermining region (CDR), or other epitope-binding fragments of anantibody.

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (“scFv”); see, e.g., Bird et al.,Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci.85:5879-5883, 1988). Such single chain antibodies are also intended tobe encompassed within the term “antigen binding fragment.” These antigenbinding fragments are obtained using conventional techniques known tothose of skill in the art, and the fragments are screened for utility inthe same manner as are intact antibodies.

Antigen binding fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies,triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger andHudson, Nature Biotechnology 23:1126-1136, 2005). Antigen bindingfragments can be grafted into scaffolds based on polypeptides such asfibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describesfibronectin polypeptide monobodies).

Antigen binding fragments can be incorporated into single chainmolecules comprising a pair of tandem Fv segments (VH—CH1-VH—CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions (Zapata et al., Protein Eng. 8:1057-1062, 1995;and U.S. Pat. No. 5,641,870).

The term “monoclonal antibody” or “monoclonal antibody composition” asused herein refers to polypeptides, including antibodies and antigenbinding fragments that have substantially identical amino acid sequenceor are derived from the same genetic source. This term also includespreparations of antibody molecules of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope.

The term “human antibody”, as used herein, includes antibodies havingvariable regions in which both the framework and CDR regions are derivedfrom sequences of human origin. Furthermore, if the antibody contains aconstant region, the constant region also is derived from such humansequences, e.g., human germline sequences, or mutated versions of humangermline sequences or antibody containing consensus framework sequencesderived from human framework sequences analysis, for example, asdescribed in Knappik et al., J. Mol. Biol. 296:57-86, 2000).

The human antibodies of the invention may include amino acid residuesnot encoded by human sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo, or aconservative substitution to promote stability or manufacturing).

The term “recognize” as used herein refers to an antibody or antigenbinding fragment thereof that finds and interacts (e.g., binds) with itsepitope, whether that epitope is linear or conformational. The term“epitope” refers to a site on an antigen to which an antibody or antigenbinding fragment of the invention specifically binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods of determining spatial conformationof epitopes include techniques in the art, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

The term “affinity” as used herein refers to the strength of interactionbetween antibody and antigen at single antigenic sites. Within eachantigenic site, the variable region of the antibody “arm” interactsthrough weak non-covalent forces with antigen at numerous sites; themore interactions, the stronger the affinity.

The term “isolated antibody” refers to an antibody that is substantiallyfree of other antibodies having different antigenic specificities. Anisolated antibody that specifically binds to one antigen may, however,have cross-reactivity to other antigens. Moreover, an isolated antibodymay be substantially free of other cellular material and/or chemicals.

The term “corresponding human germline sequence” refers to the nucleicacid sequence encoding a human variable region amino acid sequence orsubsequence that shares the highest determined amino acid sequenceidentity with a reference variable region amino acid sequence orsubsequence in comparison to all other all other known variable regionamino acid sequences encoded by human germline immunoglobulin variableregion sequences. The corresponding human germline sequence can alsorefer to the human variable region amino acid sequence or subsequencewith the highest amino acid sequence identity with a reference variableregion amino acid sequence or subsequence in comparison to all otherevaluated variable region amino acid sequences. The corresponding humangermline sequence can be framework regions only, complementaritydetermining regions only, framework and complementarity determiningregions, a variable segment (as defined above), or other combinations ofsequences or subsequences that comprise a variable region. Sequenceidentity can be determined using the methods described herein, forexample, aligning two sequences using BLAST, ALIGN, or another alignmentalgorithm known in the art. The corresponding human germline nucleicacid or amino acid sequence can have at least about 90%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the referencevariable region nucleic acid or amino acid sequence. Corresponding humangermline sequences can be determined, for example, through the publiclyavailable international ImMunoGeneTics database (IMGT) (on the worldwideweb at imgt.cines.fr/) and V-base (on the worldwide web atvbase.mrc-cpe.cam.ac.uk).

The phrase “specifically binds” or “selectively binds,” when used in thecontext of describing the interaction between an antigen (e.g., aprotein) and an antibody, antibody fragment, or antibody-derived bindingagent, refers to a binding reaction that is determinative of thepresence of the antigen in a heterogeneous population of proteins andother biologics, e.g., in a biological sample, e.g., a blood, serum,plasma or tissue sample. Thus, under certain designated immunoassayconditions, the antibodies or binding agents with a particular bindingspecificity bind to a particular antigen at least two times thebackground and do not substantially bind in a significant amount toother antigens present in the sample. In one embodiment, underdesignated immunoassay conditions, the antibody or binding agent with aparticular binding specificity binds to a particular antigen at leastten (10) times the background and does not substantially bind in asignificant amount to other antigens present in the sample. Specificbinding to an antibody or binding agent under such conditions mayrequire the antibody or agent to have been selected for its specificityfor a particular protein. As desired or appropriate, this selection maybe achieved by subtracting out antibodies that cross-react withmolecules from other species (e.g., mouse or rat) or other subtypes.Alternatively, in some embodiments, antibodies or antibody fragments areselected that cross-react with certain desired molecules.

A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Using Antibodies, A Laboratory Manual (1998), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity). Typically a specific or selective bindingreaction will produce a signal at least twice over the background signaland more typically at least 10 to 100 times over the background.

The term “equilibrium dissociation constant (KD, M)” refers to thedissociation rate constant (kd, time-1) divided by the association rateconstant (ka, time-1, M-1). Equilibrium dissociation constants can bemeasured using any known method in the art. The antibodies of thepresent invention generally will have an equilibrium dissociationconstant of less than about 10⁻⁷ or 10⁻⁸ M, for example, less than about10⁻⁹ M or 10⁻¹⁰ M, in some embodiments, less than about 10⁻¹¹ M, 10⁻¹2 Mor 10⁻¹³ M.

The term “bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

As used herein, the phrase “consisting essentially of” refers to thegenera or species of active pharmaceutical agents included in a methodor composition, as well as any excipients inactive for the intendedpurpose of the methods or compositions. In some embodiments, the phrase“consisting essentially of” expressly excludes the inclusion of one ormore additional active agents other than an antibody drug conjugate ofthe invention. In some embodiments, the phrase “consisting essentiallyof” expressly excludes the inclusion of one or more additional activeagents other than an antibody drug conjugate of the invention and asecond co-administered agent.

The term “amino acid” refers to naturally occurring, synthetic, andunnatural amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally occurring amino acids are those encoded by thegenetic code, as well as those amino acids that are later modified,e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refer to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., an α-carbon that isbound to a hydrogen, a carboxyl group, an amino group, and an R group,e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). In someembodiments, the term “conservative sequence modifications” are used torefer to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence.

The term “optimized” as used herein refers to a nucleotide sequence thathas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a yeast cell, a Pichia cell, a fungal cell, aTrichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell.The optimized nucleotide sequence is engineered to retain completely oras much as possible the amino acid sequence originally encoded by thestarting nucleotide sequence, which is also known as the “parental”sequence.

The terms “percent identical” or “percent identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refers to the extentto which two or more sequences or subsequences that are the same. Twosequences are “identical” if they have the same sequence of amino acidsor nucleotides over the region being compared. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 30 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Brent et al., Current Protocols in Molecular Biology, 2003).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller, Comput. Appl.Biosci. 4:11-17, 1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch, J. Mol. Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,(1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem.260:2605-2608; and Rossolini et al., (1994) Mol. Cell. Probes 8:91-98).

The term “operably linked” in the context of nucleic acids refers to afunctional relationship between two or more polynucleotide (e.g., DNA)segments. Typically, it refers to the functional relationship of atranscriptional regulatory sequence to a transcribed sequence. Forexample, a promoter or enhancer sequence is operably linked to a codingsequence if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “immunoconjugate” or “antibody drug conjugate” as used hereinrefers to the linkage of an antibody or an antigen binding fragmentthereof with another agent, such as a chemotherapeutic agent, a toxin,an immunotherapeutic agent, an imaging probe, and the like. The linkagecan be covalent bonds, or non-covalent interactions such as throughelectrostatic forces. Various linkers, known in the art, can be employedin order to form the immunoconjugate. Additionally, the immunoconjugatecan be provided in the form of a fusion protein that may be expressedfrom a polynucleotide encoding the immunoconjugate. As used herein,“fusion protein” refers to proteins created through the joining of twoor more genes or gene fragments which originally coded for separateproteins (including peptides and polypeptides). Translation of thefusion gene results in a single protein with functional propertiesderived from each of the original proteins.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, and reptiles.Except when noted, the terms “patient” or “subject” are used hereininterchangeably.

The term “cytotoxin”, or “cytotoxic agent” as used herein, refers to anyagent that is detrimental to the growth and proliferation of cells andmay act to reduce, inhibit, or destroy a cell or malignancy.

The term “anti-cancer agent” as used herein refers to any agent that canbe used to treat a cell proliferative disorder such as cancer, includingbut not limited to, cytotoxic agents, chemotherapeutic agents,radiotherapy and radiotherapeutic agents, targeted anti-cancer agents,and immunotherapeutic agents.

The term “drug moiety” or “payload” as used herein refers to a chemicalmoiety htat is conjugated to an antibody or antigen binding fragment ofthe invention, and can include any therapeutic or diagnostic agent, forexample, an anti-cancer, anti-inflammatory, anti-infective (e.g.,anti-fungal, antibacterial, anti-parasitic, anti-viral), or ananesthetic agent. For example, the drug moiety can be an anti-canceragent, such as a cytotoxin. In certain embodiments, a drug moiety isselected from a V-ATPase inhibitor, a HSP90 inhibitor, an IAP inhibitor,an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer,an auristatin, a dolastatin, a maytansinoid, a MetAP (methionineaminopeptidase), an inhibitor of nuclear export of proteins CRM1, aDPPIV inhibitor, an inhibitor of phosphoryl transfer reactions inmitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesininhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylatingagent, a DNA intercalator, a DNA minor groove binder and a DHFRinhibitor. Methods for attaching each of these to a linker compatiblewith the antibodies and method of the invention are known in the art.See, e.g., Singh et al., (2009) Therapeutic Antibodies: Methods andProtocols, vol. 525, 445-457. In addition, a payload can be abiophysical probe, a fluorophore, a spin label, an infrared probe, anaffinity probe, a chelator, a spectroscopic probe, a radioactive probe,a lipid molecule, a polyethylene glycol, a polymer, a spin label, DNA,RNA, a protein, a peptide, a surface, an antibody, an antibody fragment,a nanoparticle, a quantum dot, a liposome, a PLGA particle, a saccharideor a polysaccharide.

The term “maytansinoid drug moiety” means the substructure of anantibody-drug conjugate that has the structure of a maytansinoidcompound. Maytansine was first isolated from the east African shrubMaytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and maytansinol analogues have been reported. SeeU.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814;4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946;4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866;4,424,219; 4,450,254; 4,362,663; and 4,371,533, and Kawai et al (1984)Chem. Pharm. Bull. 3441-3451), each of which are expressly incorporatedby reference. Examples of specific maytansinoids useful for conjugationinclude DM1, DM3 and DM4.

“Tumor” refers to neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

The term “anti-tumor activity” means a reduction in the rate of tumorcell proliferation, viability, or metastatic activity. For example,anti-tumor activity can be shown by a decline in growth rate of abnormalcells that arises during therapy or tumor size stability or reduction,or longer survival due to therapy as compared to control withouttherapy. Such activity can be assessed using accepted in vitro or invivo tumor models, including but not limited to xenograft models,allograft models, MMTV models, and other known models known in the artto investigate anti-tumor activity.

The term “malignancy” refers to a non-benign tumor or a cancer. As usedherein, the term “cancer” includes a malignancy characterized byderegulated or uncontrolled cell growth. Exemplary cancers include:carcinomas, sarcomas, leukemias, and lymphomas.

The term “cancer” includes primary malignant tumors (e.g., those whosecells have not migrated to sites in the subject's body other than thesite of the original tumor) and secondary malignant tumors (e.g., thosearising from metastasis, the migration of tumor cells to secondary sitesthat are different from the site of the original tumor).

The term “FGFR2” refers to fibroblast growth factor receptor 2 that is amember of the receptor tyrosine kinase superfamily. The nucleic acid andamino acid sequences of FGFR2 are known, and have been published inGenBank Accession Nos. NM_000141.4, NM_001144913.1, NM_001144914.1,NM_001144915.1, NM_001144916.1, NM_001144917.1, NM_001144918.1,NM_001144919.1, NM_022970.3, NM_023029.2. Structurally, a FGFR2 aminoacid sequence is a receptor tyrosine kinase protein having a signalpeptide that is cleaved off, at least one or more immunoglobulin(Ig)-like domains, an acidic box, a transmembrane domain, and a splittyrosine kinase domain and has over its full length at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the amino acid sequence of GenBank accession numbers NM_000141.4,NM_001144913.1, NM_001144914.1, NM001144915.1, NM_001144916.1,NM_001144917.1, NM_001144918.1, NM001144919.1, NM_022970.3, NM_023029.2.Structurally, a FGFR2 nucleic acid sequence has over its full length atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity with the nucleic acid sequence of GenBank accessionnumbers NM_000141.4, NM_001144913.1, NM_001144914.1, NM_001144915.1,NM_001144916.1, NM_001144917.1, NM_001144918.1, NM_001144919.1,NM_022970.3, NM_023029.2.

The term “FGFR4” refers to fibroblast growth factor receptor 4 that is amember of the receptor tyrosine kinase superfamily. The nucleic acid andamino acid sequences of FGFR4 are known, and have been published inGenBank Accession Nos. NM_002011.3, NM_022963.2, NM_213647.1.Structurally, a FGFR4 amino acid sequence is a receptor tyrosine kinaseprotein having a signal peptide that is cleaved off, threeimmunoglobulin (Ig)-like domains, an acidic box, a transmembrane domain,and a split tyrosine kinase domain and has over its full length at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity with the amino acid sequence of GenBank accession numbersNM_002011.3, NM_022963.2, NM_213647.1. Structurally, a FGFR4 nucleicacid sequence has over its full length at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with thenucleic acid sequence of GenBank accession numbers NM_002011.3,NM_022963.2, NM213647.1.

The terms “FGFR2 expressing cancer” or “FGFR2 positive cancer” refers toa cancer that express FGFR2 or a mutation form of FGFR2 on the surfaceof cancer cells. The terms “FGFR4 expressing cancer” or “FGFR4 positivecancer” refers to a cancer that express FGFR4 or a mutation form ofFGFR4 on the surface of cancer cells.

As used herein, the terms “treat,” “treating,” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment, “treat,” “treating,” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat,” “treating,” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat,” “treating,” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

The term “therapeutically acceptable amount” or “therapeuticallyeffective dose” interchangeably refers to an amount sufficient to effectthe desired result (i.e., a reduction in tumor size, inhibition of tumorgrowth, prevention of metastasis, inhibition or prevention of viral,bacterial, fungal or parasitic infection). In some embodiments, atherapeutically acceptable amount does not induce or cause undesirableside effects. In some embodiments, a therapeutically acceptable amountinduces or causes side effects but only those that are acceptable by thehealthcare providers in view of a patient's condition. A therapeuticallyacceptable amount can be determined by first administering a low dose,and then incrementally increasing that dose until the desired effect isachieved. A “prophylactically effective dosage,” and a “therapeuticallyeffective dosage,” of the molecules of the invention can prevent theonset of, or result in a decrease in severity of, respectively, diseasesymptoms, including symptoms associated with cancer.

The term “co-administer” refers to the presence of two active agents inthe blood of an individual. Active agents that are co-administered canbe concurrently or sequentially delivered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows affinity estimates for a panel of antibodies to recombinanthuman FGFR2 IIIb.

FIG. 2 (A)-(B) show the ability of anti-FGFR antibodies to act asagonists in a Baf-FGFR2 receptor system.

FIG. 3 (A)-(B) show the ability of anti-FGFR antibodies to act asantagonists in a Baf-FGFR2 receptor system;

FIG. 4 (A)-(F), (A)-(B) show the anti-tumor activity of anti-FGFR2- andanti-FGFR2/4-MCC-DM1 ADCs in a SNU16 tumor xenograft mouse model; (C)shows the anti-tumor activity of the 12433 antibody as an ADC conjugatedto SMCC-DM1 and SPDB-DM4 linker-payloads; (D)-(E) show thepharmacokinetic properties of anti-FGFR2- and anti-FGFR2/4-MCC-DM1 ADCs;(F) shows the anti-tumor activity against ADC clearance for a panel ofanti-FGFR2- and anti-FGFR2/4-MCC-DM1 ADCs.

FIG. 5 (A)-(C) are western blots showing the ability of anti-FGFRantibodies or ADCs to modulate FGFR signaling and total receptorexpression in FGFR2-amplified cell lines, SNU16 after 2 hours (A), 2-72hours (B) or Kato-III cells (C).

FIG. 6 (A)-(D) show the ability of 12425-MCC-DM1 to inhibit theproliferation of SNU-16 (A), Kato-III (B) or NUGC3 (C) cells relative toIgG and free payload controls; (D) is shows that the unconjugatedantibody, 12425, did not have any appreciable anti-proliferative effect.

FIG. 7 (A)-(B) are images of SNU-16 (A) or NUGC3 (B) tumor xenograftsfollowing treatment with 12425-MCC-DM1 that show the assessment of pHH3and cleaved caspase 3 expression.

FIG. 8 (A)-(C) show the anti-tumor activity of anti-FGFR2- andanti-FGFR2/4-MCC-DM1 ADCs in H716 (A), MFM223 (B) and CHGA-010 (C) tumorxenograft mouse models.

FIG. 9 shows the anti-tumor activity of the anti FGFR2/4 ADC,12425-MCC-DM1, alone and in combination with the FGFR tyrosine kinaseinhibitor, BGJ398 in the CHGA-119 tumor xenograft model.

FIG. 10 shows the anti-tumor activity of the anti FGFR2/4 ADC,12425-MCC-DM1 at different doses and schedules in the SNU-16 tumorxenograft mouse model.

FIG. 11 (A)-(D), (A)-(C) show the assessment of the the ability ofanti-FGFR2 antibodies to induce ADCC in vitro in Kato III cells (A), tobind to C1q (B), induce CDC in Kato III cells. (D) shows the effect ofan ADCC depleted variant as an unconjugated antibody or MCC-DM1conjugated ADC in vivo.

FIG. 12 (A)-(B) are western blots showing the ability of anti-FGFRantibodies or ADCs to modulate FGFR signaling and total receptorexpression in the FGFR4-expressing cell line, MDA-MB453 after 2 hours(A) or over a time course (B).

FIG. 13 (A)-(D) show the ability of FGFR2 or FGFR2/4 antibodies(unconjugated or as MCC-DM1 ADCs) to inhibit the proliferation MDA-MB453(A), RH4 (B), JR cells (C) or a cancer cell line panel (D).

FIG. 14 (A)-(C) are images of MDA-MB453 ((A) and (B)) or RH4 (C) tumorxenografts following treatment with 12425-MCC-DM1 that show theassessment of pHH3 and/or cleaved caspase 3 expression.

FIG. 15 (A)-(B) show the anti-tumor activity of anti-FGFR2- andanti-FGFR2/4-MCC-DM1 ADCs in MDA-MB453 (A), and RH4 (B) tumor xenograftmouse models.

FIG. 16 (A)-(D) show the PK profiles of 12425-MCC-DM1 in mouse((A)-(B)), rat (C), or cynomolgus monkey (D).

FIG. 17 (A)-(B) show the ability of the engineered variants of 12425(20562-MCC-DM1) to inhibit the proliferation of SNU-16 cells in vitro(A) and in vivo (B).

FIG. 18 (A)-(B) show the ability of the affinity-matured variants of10164 (20809-MCC-DM1) and 12425 (20811-MCC-DM1) to inhibit theproliferation of SNU-16 cells in vitro (A) and in vivo (B).

FIG. 19 (A)-(D) shows the results of deuterium exchange experiments toFGFR2. (A) shows the absolute deuterium uptake for FGFR2 in the absence(control) and presence of six therapeutic mAb. The heights of the barsare the average of three measurements and the error bars are onestandard deviation; (B) shows the difference in deuterium uptake betweenmAb bound and control FGFR2 divided by the standard error in themeasurement; (C) shows regions of high protection upon binding of mAbsto FGFR2 mapped onto FGFR2 IIIc: FGFR2 crystal structure (PDB ID: 1EV2);and (D) shows regions of high protection upon binding of mAbs to FGFR2mapped onto FGFR2 IIIb: FGFR1 crystal structure (PDB ID: 3OJM).

FIG. 20 (A)-(D) shows the results from X-ray crystallography epitopemapping studies. (A) shows the overall structure of 12425 Fab binding toFGFR2 (left panel) and detailed interaction surface on FGFR2 withepitope residues labeled (right panel) (figure discloses residues atindicated positions of SEQ ID NO: 137); (B)-(C) show two dimerizationmodels of FGFR-FGF-heparin signaling complex (upper panels) and theclash of 12425 Fab with both models (lower panels); and (D) shows asequence alignment of human FGFR2 (SEQ ID NO: 145) and FGFR4 (SEQ ID NO:146) in the D2 domain. Completely conserved residues are shaded in darkgrey, partially conserved residues in light grey. Dashed boxes are FGFR2residues contacting 12425 Fab, along with their aligned counterparts inFGFR4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibodies, antibody fragments (e.g.,antigen binding fragments), and antibody drug conjugates that bind toboth FGFR2 and FGFR4 (“FGFR2/4”). In particular, the present inventionprovides antibodies and antibody fragments (e.g., antigen bindingfragments) that bind to FGFR2/4, and internalize upon such binding. Theantibodies and antibody fragments (e.g., antigen binding fragments) ofthe present invention can be used for producing antibody drugconjugates. Furthermore, the present invention provides antibody drugconjugates that have desirable pharmacokinetic charicteristics and otherdesirable attributes, and thus can be used for treating cancerexpressing FGFR2 and/or FGFR4, such as gastric cancer, breast cancer,alveolar rhabdomyosarcoma, liver cancer, adrenal cancer, lung cancer,colon cancer and endometrial cancer. The present invention furtherprovides pharmaceutical compositions comprising the antibody drugconjugates of the invention, and methods of making and using suchpharmaceutical compositions for the treatment of cancer.

Antibody Drug Coniugates

The present invention provides antibody drug conjugates, where anantibody, antigen binding fragment or its functional equivalent thatspecifically binds to FGFR2/4 is linked to a drug moiety. In one aspect,the antibodies, antigen binding fragments or their functionalequivalents of the invention are linked, via covalent attachment by alinker, to a drug moiety that is an anti-cancer agent. The antibody drugconjugates of the invention can selectively deliver an effective dose ofan anti-cancer agent (e.g., a cytotoxic agent) to tumor tissuesexpressing FGFR2 and/or FGFR4, whereby greater selectivity (and lowerefficacious dose) may be achieved.

In one aspect, the invention provides an immunoconjugate of Formula (I):

Ab-(L-(D)_(m))_(n)

Wherein Ab represents an FGFR2/4 binding antibody described herein;L is a linker;D is a drug moiety;m is an integer from 1 to 8; andn is an integer from 1-20. In one embodiment, n is an integer from 1 to10, 2 to 8, or 2 to 5. In a specific embodiment, n is 2, 3, or 4. Insome embodiments, m is 1; in other embodiments m is 2, 3 or 4.

While the drug to antibody ratio has an exact value for a specificconjugate molecule (e.g., n multiplied by m in Formula (I)), it isunderstood that the value will often be an average value when used todescribe a sample containing many molecules, due to some degree ofinhomogeneity, typically associated with the conjugation step. Theaverage loading for a sample of an immunoconjugate is referred to hereinas the drug to antibody ratio, or “DAR.” In some embodiments, when thedrug is maytansinoid, it is referred to as “MAR.” In some embodiments,the DAR is between about 1 and about 5, and typically is about 3, 3.5,4, 4.5, or 5. In some embodiments, at least 50% of a sample by weight iscompound having the average DAR plus or minus 2, and preferably at least50% of the sample is a conjugate that contains the average DAR plus orminus 1. Preferred embodiments include immunoconjugates wherein the DARis about 3.5. In some embodiments, a DAR of ‘about n’ means the measuredvalue for DAR is within 20% of n.

The present invention is also directed to immunoconjugates comprisingthe antibodies, antibody fragments (e.g., antigen binding fragments) andtheir functional equivalents as disclosed herein, linked or conjugatedto a drug moiety. In one embodiment, the drug moiety D is a maytansinoiddrug moiety, including those having the structure:

where the wavy line indicates the covalent attachment of the sulfur atomof the maytansinoid to a linker of an antibody drug conjugate. R at eachoccurrence is independently H or a C₁-C₆ alkyl. The alkylene chainattaching the amide group to the sulfur atom may be methanyl, ethanyl,or propyl, i.e. m is 1, 2, or 3. (U.S. Pat. No. 633,410, U.S. Pat. No.5,208,020, Chari et al. (1992) Cancer Res. 52; 127-131, Lui et al.(1996) Proc. Natl. Acad. Sci. 93:8618-8623).

All stereoisomers of the maytansinoid drug moiety are contemplated forthe immunoconjugates of the invention, i.e. any combination of R and Sconfigurations at the chiral carbons of the maytansinoid. In oneembodiment the maytansinoid drug moiety has the followingstereochemistry.

In one embodiment, the maytansinoid drug moiety isN^(2′)-deacetyl-N^(2′)-(3-mercapto-1-oxopropyl)-maytansine (also knownas DM1). DM1 is represented by the following structural formula.

In another embodiment the maytansinoid drug moiety isN^(2′)-deacetyl-N^(2′)-(4-mercapto-1-oxopentyl)-maytansine (also knownas DM3). DM3 is respresented by the following structural formula.

In another embodiment the maytansinoid drug moiety isN^(2′)-deacetyl-N^(2′)-(4-methyl-4-mercapto-1-oxopentyl)-maytansine(also known as DM4). DM4 is represented by the following structuralformula.

The drug moiety D can be linked to the antibody Ab through linker L. Lis any chemical moiety capable of linking the drug moiety to theantibody through covalent bonds. A cross-linking reagent is abifunctional or multifunctional reagent that can be used to link a drugmoiety and an antibody to form antibody drug conjugates. Antibody drugconjugates can be prepared using a cross-linking reagent having areactive functionality capable of binding to both the drug moiety andthe antibody. For example, a cysteine, thiol or an amine, e.g.N-terminus or an amino acid side chain, such as lysine of the antibody,can form a bond with a functional group of a cross-linking reagent.

In one embodiment, L is a cleavable linker. In another embodiment, L isa non-cleavable linker. In some embodiments, L is an acid-labile linker,photo-labile linker, peptidase cleavable linker, esterase cleavablelinker, a disulfide bond cleavable linker, a hydrophilic linker, aprocharged linker, or a dicarboxylic acid based linker.

Suitable cross-linking reagents that form a non-cleavable linker betweenthe drug moiety, for example maytansinoid, and the antibody are wellknown in the art, and can form non-cleavable linkers that comprise asulfur atom (such as SMCC) or those that are without a sulfur atom.Preferred cross-linking reagents that form non-cleavable linkers betweenthe drug moiety, for example maytansinoid, and the antibody comprise amaleimido- or haloacetyl-based moiety. According to the presentinvention, such non-cleavable linkers are said to be derived frommaleimido- or haloacetyl-based moieties.

Cross-linking reagents comprising a maleimido-based moiety include butnot limited to, N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate(SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC),N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate),which is a “long chain” analog of SMCC (LC-SMCC), K-maleimidoundeconoicacid N-succinimidyl ester (KMUA), γ-maleimidobutyric acid N-succinimidylester (GMBS), ε-maleimidocaproic acid N-succinimidyl ester (EMCS),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),N-(α-maleimidoacetoxy)-succinimide ester (AMSA),succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH),N-succinimidyl-4-(p-maleimidophenyl)-butyrate (SMPB),N-(-p-maleomidophenyl)isocyanate (PMIP) and maleimido-basedcross-linking reagents containing a polyethythene glycol spacer, such asMAL-PEG-NHS. These cross-linking reagents form non-cleavable linkersderived from maleimido-based moieties. Representative structures ofmaleimido-based cross-linking reagents are shown below.

In another embodiment, the linker L is derived fromN-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate(sulfo-SMCC) or MAL-PEG-NHS.

Cross-linking reagents comprising a haloacetyle-based moiety includeN-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidylbromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate(SBAP). These cross-linking reagents form a non-cleavable linker derivedfrom haloacetyl-based moieties. Representative structures ofhaloacetyl-based cross-linking reagents are shown below.

In one embodiment, the linker L is derived from N-succinimidyliodoacetate (SIA) or N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB).

Suitable cross-linking reagents that form a cleavable linker between thedrug moiety, for example maytansinoid, and the antibody are well knownin the art. Disulfide containing linkers are linkers cleavable throughdisulfide exchange, which can occur under physiological conditions.According to the present invention, such cleavable linkers are said tobe derived from disulfide-based moieties. Suitable disulfidecross-linking reagents includeN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) andN-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB), thestructures of which are shown below. These disulfide cross-linkingreagents form a cleavable linker derived from disulfide-based moieties.

N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),

N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP),

N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and

N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB).

In one embodiment, the linker L is derived fromN-succinimidyl-4-(2-pyridyldithio) butanoate (SPDB).

Suitable cross-linking reagents that form a charged linker between thedrug moiety, for example maytansinoid, and the antibody are known asprocharged cross-linking reagents. In one embodiment, the linker L isderived from the procharged cross-linking reagent CX1-1. The structureof CX1-1 is below.

2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1)

Each of the cross-linking reagents depicted above contains, at one endof the cross-linking reagent, a NHS-ester which reacts with a primaryamine of the antibody to form an amide bond and, at the other end, amaleimide group or pyridinyldisulfide group which reacts with thesulfhydryl of the maytansionoid drug moiety to form a thioether ordisulfide bond.

In one embodiment, the conjugate of the present invention is representedby any one of the following structural formulae

wherein:

Ab is an antibody or antigen binding fragment thereof that specificallybinds to both human FGFR2 and FGFR4;

n, which indicates the number of D-L groups attached the Ab through theformation of an amide bond with a primary amine of the Ab, is an integerfrom 1 to 20. In one embodiment, n is an integer from 1 to 10, 2 to 8 or2 to 5. In a specific embodiment, n is 3 or 4.

In one embodiment, the average molar ratio of drug (e.g., DM1 or DM4) tothe antibody in the conjugate (i.e., average n value, also known asMaytanisnoid Antibody Ratio (MAR)) is about 1 to about 10, about 2 toabout 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or 8.1), about 2.5 to about7, about 3 to about 5, about 2.5 to about 4.5 (e.g., about 2.5, about2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.3,about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5), about 3.0to about 4.0, about 3.2 to about 4.2, or about 4.5 to 5.5 (e.g., about4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, or about 5.5).

In one aspect of the invention, the conjugate of the present inventionhas substantially high purity and has one or more of the followingfeatures: (a) greater than about 90% (e.g., greater than or equal toabout 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), preferablygreater than about 95%, of conjugate species are monomeric, (b)unconjugated linker level in the conjugate preparation is less thanabout 10% (e.g., less than or equal to about 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, or 0%) (relative to total linker), (c) less than 10% ofconjugate species are crosslinked (e.g., less than or equal to about 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%), (d) free drug (e.g., DM1 or DM4)level in the conjugate preparation is less than about 2% (e.g., lessthan or equal to about 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%,0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0%) (mol/mol relative tototal cytotoxic agent) and/or (e) no substantial increase in the levelof free drug (e.g., DM1 or DM4) occurs upon storage (e.g., after about 1week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about3 months, about 4 months, about 5 months, about 6 months, about 1 year,about 2 years, about 3 years, about 4 years, or about 5 years).“Substantial increase” in the level of free drug (e.g., DM1 or DM4)means that after certain storage time (e.g., about 1 week, about 2weeks, about 3 weeks, about 1 month, about 2 months, about 3 months,about 4 months, about 5 months, about 6 months, about 1 year, about 2years, about 3 years, about 4 years, or about 5 years), the increase inthe level of free drug (e.g., DM1 or DM4) is less than about 0.1%, about0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about2.0%, about 2.2%, about 2.5%, about 2.7%, about 3.0%, about 3.2%, about3.5%, about 3.7%, or about 4.0%.

As used herein, the term “unconjugated linker” refers to the antibodythat is covalently linked with a linker derived from a cross-linkingreagent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1), wherein theantibody is not covalently coupled to the drug (e.g., DM1 or DM4)through a linker (i.e., the “unconjugated linker” can be represented byAb-MCC, Ab-SPDB, or Ab-CX1-1).

1. Drug Moiety

The present invention provides immunoconjugates that specifically bindto FGFR2/4. The immunoconjugates of the invention comprise anti-FGFR2/4antibodies, antibody fragments (e.g., antigen binding fragments) orfunctional equivalents that are conjugated to a drug moiety, e.g., ananti-cancer agent, an autoimmune treatment agent, an anti-inflammatoryagent, an antifungal agent, an antibacterial agent, an anti-parasiticagent, an anti-viral agent, or an anesthetic agent. The antibodies,antibody fragments (e.g., antigen binding fragments) or functionalequivalents of the invention can be conjugated to several identical ordifferent drug moieties using any methods known in the art.

In certain embodiments, the drug moiety of the immunoconjugates of thepresent invention is selected from a group consisting of a V-ATPaseinhibitor, a pro-apoptotic agent, a Bcl2 inhibitor, an MCL1 inhibitor, aHSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubulestabilizer, a microtubule destabilizer, an auristatin, a dolastatin, amaytansinoid, a MetAP (methionine aminopeptidase), an inhibitor ofnuclear export of proteins CRM1, a DPPIV inhibitor, proteasomeinhibitors, an inhibitor of phosphoryl transfer reactions inmitochondria, a protein synthesis inhibitor, a kinase inhibitor, a CDK2inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, aDNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNAminor groove binder and a DHFR inhibitor.

In one embodiment, the drug moiety of the immunoconjugates of thepresent invention is a maytansinoid drug moiety, such as but not limitedto, DM1, DM3, or DM4.

Further, the antibodies, antibody fragments (e.g., antigen bindingfragments) or functional equivalents of the present invention may beconjugated to a drug moiety that modifies a given biological response.Drug moieties are not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein,peptide, or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, cholera toxin, or diphtheria toxin, a protein suchas tumor necrosis factor, α-interferon, β-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator, acytokine, an apoptotic agent, an anti-angiogenic agent, or, a biologicalresponse modifier such as, for example, a lymphokine.

In one embodiment, the antibodies, antibody fragments (e.g., antigenbinding fragments) or functional equivalents of the present inventionare conjugated to a drug moiety, such as a cytotoxin, a drug (e.g., animmunosuppressant) or a radiotoxin. Examples of cytotoxins include butare not limited to, taxanes (see, e.g., International (PCT) PatentApplication Nos. WO 01/38318 and PCT/US03/02675), DNA-alkylating agents(e.g., CC-1065 analogs), anthracyclines, tubulysin analogs, duocarmycinanalogs, auristatin E, auristatin F, maytansinoids, and cytotoxic agentscomprising a reactive polyethylene glycol moiety (see, e.g., Sasse etal., J. Antibiot. (Tokyo), 53, 879-85 (2000), Suzawa et al., Bioorg.Med. Chem., 8, 2175-84 (2000), Ichimura et al., J. Antibiot. (Tokyo),44, 1045-53 (1991), Francisco et al., Blood (2003) (electronicpublication prior to print publication), U.S. Pat. Nos. 5,475,092,6,340,701, 6,372,738, and 6,436,931, U.S. Patent Application PublicationNo. 2001/0036923 A1, Pending U.S. patent application Ser. Nos.10/024,290 and 10/116,053, and International (PCT) Patent ApplicationNo. WO 01/49698), taxon, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, t.colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents alsoinclude, for example, anti-metabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), ablating agents (e.g., mechlorethamine, thiotepachlorambucil, meiphalan, carmustine (BSNU) and lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), and anti-mitotic agents (e.g., vincristine andvinblastine). (See e.g., Seattle Genetics US20090304721).

Other examples of cytotoxins that can be conjugated to the antibodies,antibody fragments (antigen binding fragments) or functional equivalentsof the invention include duocarmycins, calicheamicins, maytansines andauristatins, and derivatives thereof.

Various types of cytotoxins, linkers and methods for conjugatingtherapeutic agents to antibodies are known in the art, see, e.g., Saitoet al., (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail et al., (2003)Cancer Immunol. Immunother. 52:328-337; Payne, (2003) Cancer Cell3:207-212; Allen, (2002) Nat. Rev. Cancer 2:750-763; Pastan andKreitman, (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter andSpringer, (2001) Adv. Drug Deliv. Rev. 53:247-264.

The antibodies, antibody fragments (e.g., antigen binding fragments) orfunctional equivalents of the present invention can also be conjugatedto a radioactive isotope to generate cytotoxic radiopharmaceuticals,referred to as radioimmunoconjugates. Examples of radioactive isotopesthat can be conjugated to antibodies for use diagnostically ortherapeutically include, but are not limited to, iodine-131, indium-111,yttrium-90, and lutetium-177. Methods for preparingradioimmunoconjugates are established in the art. Examples ofradioimmunoconjugates are commercially available, including Zevalin™(DEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similarmethods can be used to prepare radioimmunoconjugates using theantibodies of the invention. In certain embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid(DOTA) which can be attached to the antibody via a linker molecule. Suchlinker molecules are commonly known in the art and described in Denardoet al., (1998) Clin Cancer Res. 4(10):2483-90; Peterson et al., (1999)Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., (1999) Nucl. Med.Biol. 26(8):943-50, each incorporated by reference in their entireties.

The antibodies, antibody fragments (e.g., antigen binding fragments) orfunctional equivalents of the present invention can also conjugated to aheterologous protein or polypeptide (or fragment thereof, preferably toa polypeptide of at least 10, at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, at least 80, at least 90 or at least100 amino acids) to generate fusion proteins. In particular, theinvention provides fusion proteins comprising an antibody fragment(e.g., antigen binding fragment) described herein (e.g., a Fab fragment,Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VLdomain or a VL CDR) and a heterologous protein, polypeptide, or peptide.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama, (1998) TrendsBiotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol.287:265-76; and Lorenzo and Blasco, (1998) Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody or fragment thereof that specifically binds to anantigen may be recombined with one or more components, motifs, sections,parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies, antibody fragments (e.g., antigen bindingfragments) or functional equivalents of the present invention can beconjugated to marker sequences, such as a peptide, to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide (SEQ ID NO: 142), such as the tag providedin a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311), among others, many of which are commercially available. Asdescribed in Gentz et al., (1989) Proc. Natl. Acad. Sci. USA 86:821-824,for instance, hexa-histidine (SEQ ID NO: 142) provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the hemagglutinin (“HA”)tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., (1984) Cell 37:767), and the“FLAG” tag (A. Einhauer et al., J. Biochem. Biophys. Methods 49:455-465, 2001). According to the present invention, antibodies orantigen binding fragments can also be conjugated to tumor-penetratingpeptides in order to enhance their efficacy.

In other embodiments, the antibodies, antibody fragments (e.g., antigenbinding fragments) or functional equivalents of the present inventionare conjugated to a diagnostic or detectable agent. Suchimmunoconjugates can be useful for monitoring or prognosing the onset,development, progression and/or severity of a disease or disorder aspart of a clinical testing procedure, such as determining the efficacyof a particular therapy. Such diagnosis and detection can beaccomplished by coupling the antibody to detectable substancesincluding, but not limited to, various enzymes, such as, but not limitedto, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials, such as,but not limited to, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532,Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660,Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as, but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as, but not limitedto, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In), technetium (⁹⁹Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ⁶⁴Cu, ¹¹³Sn, and¹¹⁷Sn; and positron emitting metals using various positron emissiontomographies, and non-radioactive paramagnetic metal ions.

The antibodies, antibody fragments (e.g., antigen binding fragments) orfunctional equivalents of the invention may also be attached to solidsupports, which are particularly useful for immunoassays or purificationof the target antigen. Such solid supports include, but are not limitedto, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinylchloride or polypropylene.

2. Linker

As used herein, a “linker” is any chemical moiety that is capable oflinking an antibody, antibody fragment (e.g., antigen binding fragments)or functional equivalent to another moiety, such as a drug moeity.Linkers can be susceptible to cleavage (cleavable linker), such as,acid-induced cleavage, photo-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage, and disulfide bond cleavage, atconditions under which the compound or the antibody remains active.Alternatively, linkers can be substantially resistant to cleavage (e.g.,stable linker or noncleavable linker). In some aspects, the linker is aprocharged linker, a hydrophilic linker, or a dicarboxylic acid basedlinker.

In one aspect, the linker used in the present invention is derived froma crosslinking reagent such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1). In another aspect, the linker used in the present invention isderived from a cross-linking agent such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimidyl4-(maleimidomethyl) cyclohexanecarboxylate (sulfo-SMCC),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB) or2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1).

Non-cleavable linkers are any chemical moiety capable of linking a drug,such as a maytansinoid, to an antibody in a stable, covalent manner anddoes not fall off under the categorties listed above for cleaveablelinkers. Thus, non-cleavable linkers are substantialy resistant toacid-induced cleavage, photo-induced cleavage, peptidase-inducedcleavage, esterase-induced cleavage and disulfide bond cleavage.Furthermore, non-cleavable refers to the ability of the chemical bond inthe linker or adjoining to the linker to withstand cleavage induced byan acid, photolabile-cleaving agent, a peptidase, an esterase, or achemical or physiological compound that cleaves a disulfide bond, atconditions under which the drug, such as maytansionoid or the antibodydoes not lose its activity.

Acid-labile linkers are linkers cleavable at acidic pH. For example,certain intracellular compartments, such as endosomes and lysosomes,have an acidic pH (pH 4-5), and provide conditions suitable to cleaveacid-labile linkers.

Photo-labile linkers are linkers that are useful at the body surface andin many body cavities that are accessable to light. Furthermore,infrared light can penetrate tissue.

Some linkers can be cleaved by peptidases, i.e. peptidase cleavablelinkers. Only certain peptides are readily cleaved inside or outsidecells, see e.g. Trout et al., 79 Proc. Natl. Acad. Sci. USA, 626-629(1982) and Umemoto et al. 43 Int. J. Cancer, 677-684 (1989).Furthermore, peptides are composed of α-amino acids and peptidic bonds,which chemically are amide bonds between the carboxylate of one aminoacid and the amino group of a second amino acid. Other amide bonds, suchas the bond between a carboxylate and the ε-amino group of lysine, areunderstood not to be peptidic bonds and are considered non-cleavable.

Some linkers can be cleaved by esterases, i.e. esterase cleavablelinkers. Again, only certain esters can be cleaved by esterases presentinside or outside of cells. Esters are formed by the condensation of acarboxylic acid and an alcohol. Simple esters are esters produced withsimple alcohols, such as aliphatic alcohols, and small cyclic and smallaromatic alcohols.

Procharged linkers are derived from charged cross-linking reagents thatretain their charge after incorporation into an antibody drug conjugate.Examples of procharged linkers can be found in US 2009/0274713.

3. Conjugation and Preparation of ADCs

The conjugates of the present invention can be prepared by any methodsknown in the art, such as those described in U.S. Pat. Nos. 7,811,572,6,411,163, 7,368,565, and 8,163,888, and US application publications2011/0003969, 2011/0166319, 2012/0253021 and 2012/0259100. The entireteachings of these patents and patent application publications areherein incorporated by reference.

One-Step Process

In one embodiment, the conjugates of the present invention can beprepared by a one-step process. The process comprises combining theantibody, drug and cross-linking agent in a substantially aqueousmedium, optionally containing one or more co-solvents, at a suitable pH.In one embodiment, the process comprises the step of contacting theantibody of the present invention with a drug (e.g., DM1 or DM4) to forma first mixture comprising the antibody and the drug, and thencontacting the first mixture comprising the antibody and the drug with across-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1)in a solution having a pH of about 4 to about 9 to provide a mixturecomprising (i) the conjugate (e.g., Ab-MCC-DM1, Ab-SPDB-DM4, orAb-CX1-1-DM1), (ii) free drug (e.g., DM1 or DM4), and (iii) reactionby-products.

In one embodiment, the one-step process comprises contacting theantibody with the drug (e.g., DM1 or DM4) and then the cross-linkingagent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) in a solutionhaving a pH of about 6 or greater (e.g., about 6 to about 9, about 6 toabout 7, about 7 to about 9, about 7 to about 8.5, about 7.5 to about8.5, about 7.5 to about 8.0, about 8.0 to about 9.0, or about 8.5 toabout 9.0). For example, the inventive process comprises contacting acell-binding agent with the drug (DM1 or DM4) and then the cross-linkingagent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) in a solutionhaving a pH of about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7,about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about9.0. In a specific embodiment, the inventive process comprisescontacting a cell-binding agent with the drug (e.g., DM1 or DM4) andthen the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDBor CX1-1) in a solution having a pH of about 7.8 (e.g., a pH of 7.6 to8.0 or a pH of 7.7 to 7.9).

The one-step process (i.e., contacting the antibody with the drug (e.g.,DM1 or DM4) and then the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1) can be carried out at any suitabletemperature known in the art. For example, the one-step process canoccur at about 20° C. or less (e.g., about −10° C. (provided that thesolution is prevented from freezing, e.g., by the presence of organicsolvent used to dissolve the cytotoxic agent and the bifunctionalcrosslinking reagent) to about 20° C., about 0° C. to about 18° C.,about 4° C. to about 16° C.), at room temperature (e.g., about 20° C. toabout 30° C. or about 20° C. to about 25° C.), or at an elevatedtemperature (e.g., about 30° C. to about 37° C.). In one embodiment, theone-step process occurs at a temperature of about 16° C. to about 24° C.(e.g., about 16° C., about 17° C., about 18° C., about 19° C., about 20°C., about 21° C., about 22° C., about 23° C., about 24° C., or about 25°C.). In another embodiment, the one-step process is carried out at atemperature of about 15° C. or less (e.g., about −10° C. to about 15°C., or about 0° C. to about 15° C.). For example, the process comprisescontacting the antibody with the drug (e.g., DM1 or DM4) and then thecross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1)at a temperature of about 15° C., about 14° C., about 13° C., about 12°C., about 11° C., about 10° C., about 9° C., about 8° C., about 7° C.,about 6° C., about 5° C., about 4° C., about 3° C., about 2° C., about1° C., about 0° C., about −1° C., about −2° C., about −3° C., about −4°C., about −5° C., about −6° C., about −7° C., about −8° C., about −9°C., or about −10° C., provided that the solution is prevented fromfreezing, e.g., by the presence of organic solvent(s) used to dissolvethe cross-linking agent (e.g., SMCC, Sulfo-SMCC, Sulfo-SPDB SPDB, orCX1-1). In one embodiment, the process comprises contacting the antibodywith the drug (e.g., DM1 or DM4) and then the cross-linking agent (e.g.,SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) at a temperature of about−10° C. to about 15° C., about 0° C. to about 15° C., about 0° C. toabout 10° C., about 0° C. to about 5° C., about 5° C. to about 15° C.,about 10° C. to about 15° C., or about 5° C. to about 10° C. In anotherembodiment, the process comprises contacting the antibody with the drug(e.g., DM1 or DM4) and then the cross-linking agent (e.g., SMCC,Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) at a temperature of about 10° C.(e.g., a temperature of 8° C. to 12° C. or a temperature of 9° C. to 11°C.).

In one embodiment, the contacting described above is effected byproviding the antibody, then contacting the antibody with the drug(e.g., DM1 or DM4) to form a first mixture comprising the antibody andthe drug (e.g., DM1 or DM4), and then contacting the first mixturecomprising the antibody and the drug (e.g., DM1 or DM4) with thecross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).For example, in one embodiment, the antibody is provided in a reactionvessel, the drug (e.g., DM1 or DM4) is added to the reaction vessel(thereby contacting the antibody), and then the cross-linking agent(e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) is added to themixture comprising the antibody and the drug (e.g., DM1 or DM4) (therebycontacting the mixture comprising the antibody and the drug). In oneembodiment, the antibody is provided in a reaction vessel, and the drug(e.g., DM1 or DM4) is added to the reaction vessel immediately followingproviding the antibody to the vessel. In another embodiment, theantibody is provided in a reaction vessel, and the drug (e.g., DM1 orDM4) is added to the reaction vessel after a time interval followingproviding the antibody to the vessel (e.g., about 5 minutes, about 10minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 1 hour, about 1 day or longer after providing thecell-binding agent to the space). The drug (e.g., DM1 or DM4) can beadded quickly (i.e., within a short time interval, such as about 5minutes, about 10 minutes) or slowly (such as by using a pump).

The mixture comprising the antibody and the drug (e.g., DM1 or DM4) canthen be contacted with the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1) either immediately after contacting theantibody with the drug (e.g., DM1 or DM4) or at some later point (e.g.,about 5 minutes to about 8 hours or longer) after contacting theantibody with the drug (e.g., DM1 or DM4). For example, in oneembodiment, the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB,Sulfo-SPDB or CX1-1) is added to the mixture comprising the antibody andthe drug (e.g., DM1 or DM4) immediately after the addition of the drug(e.g., DM1 or DM4) to the reaction vessel comprising the antibody.Alternatively, the mixture comprising the antibody and the drug (e.g.,DM1 or DM4) can be contacted with the cross-linking agent (e.g., SMCC,Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1) at about 5 minutes, about 10minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about7 hours, about 8 hours, or longer after contacting the antibody with thedrug (e.g., DM1 or DM4).

After the mixture comprising the antibody and the drug (e.g., DM1 orDM4) is contacted with the cross-linking agent (e.g., SMCC, Sulfo-SMCC,SPDB, Sulfo-SPDB or CX1-1) the reaction is allowed to proceed for about1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours,about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours, or longer (e.g., about 30 hours, about 35 hours, about40 hours, about 45 hours, or about 48 hrs).

In one embodiment, the one-step process further comprises a quenchingstep to quench any unreacted drug (e.g., DM1 or DM4) and/or unreactedcross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).The quenching step is typically performed prior to purification of theconjugate. In one embodiment, the mixture is quenched by contacting themixture with a quenching reagent. As used herein, the “quenchingreagent” refers to a reagent that reacts with the free drug (e.g., DM1or DM4) and/or cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB,Sulfo-SPDB or CX1-1). In one embodiment, maleimide or haloacetamidequenching reagents, such as 4-maleimidobutyric acid,3-maleimidopropionic acid, N-ethylmaleimide, iodoacetamide, oriodoacetamidopropionic acid, can be used to ensure that any unreactedgroup (such as thiol) in the drug (e.g., DM1 or DM4) is quenched. Thequenching step can help prevent the dimerization of the drug (e.g.,DM1). The dimerized DM1 can be difficult to remove. Upon quenching withpolar, charged thiol-quenching reagents (such as 4-maleimidobutyric acidor 3-maleimidopropionic acid), the excess, unreacted DM1 is convertedinto a polar, charged, water-soluble adduct that can be easily separatedfrom the covalently-linked conjugate during the purification step.Quenching with non-polar and neutral thiol-quenching reagents can alsobe used. In one embodiment, the mixture is quenched by contacting themixture with a quenching reagent that reacts with the unreactedcross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).For example, nucleophiles can be added to the mixture in order to quenchany unreacted SMCC. The nucleophile preferably is an amino groupcontaining nucleophile, such as lysine, taurine and hydroxylamine.

In a preferred embodiment, the reaction (i.e., contacting the antibodywith the drug (e.g., DM1 or DM4) and then cross-linking agent (e.g.,SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1)) is allowed to proceed tocompletion prior to contacting the mixture with a quenching reagent. Inthis regard, the quenching reagent is added to the mixture about 1 hourto about 48 hours (e.g., about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours,about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours,about 22 hours, about 23 hours, about 24 hours, or about 25 hours toabout 48 hours) after the mixture comprising the antibody and the drug(e.g., DM1 or DM4) is contacted with the cross-linking agent (e.g.,SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).

Alternatively, the mixture is quenched by lowering the pH of the mixtureto about 5.0 (e.g., 4.8, 4.9, 5.0, 5.1 or 5.2). In another embodiment,the mixture is quenched by lowering the pH to less than 6.0, less than5.5, less than 5.0, less than 4.8, less than 4.6, less than 4.4, lessthan 4.2, less than 4.0. Alternatively, the pH is lowered to about 4.0(e.g., 3.8, 3.9, 4.0, 4.1 or 4.2) to about 6.0 (e.g., 5.8, 5.9, 6.0, 6.1or 6.2), about 4.0 to about 5.0, about 4.5 (e.g., 4.3, 4.4, 4.5, 4.6 or4.7) to about 5.0. In one embodiment, the mixture is quenched bylowering the pH of the mixture to 4.8. In another embodiment, themixture is quenched by lowering the pH of the mixture to 5.5.

In one embodiment, the one-step process further comprises a holding stepto release the unstably bound linkers from the antibody. The holdingstep comprises holding the mixture prior to purification of theconjugate (e.g., after the reaction step, between the reaction step andthe quenching step, or after the quenching step). For example, theprocess comprises (a) contacting the antibody with the drug (e.g., DM1or DM4) to form a mixture comprising the antibody and the drug (e.g.,DM1 or DM4); and then contacting the mixture comprising the antibody anddrug (e.g., DM1 or DM4) with the cross-linking agent (e.g., SMCC,Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1), in a solution having a pH ofabout 4 to about 9 to provide a mixture comprising (i) the conjugate(e.g., Ab-MCC-DM1, Ab-SPDB-DM4 or Ab-CX1-1-DM1), (ii) free drug (e.g.,DM1 or DM4), and (iii) reaction by-products, (b) holding the mixtureprepared in step (a) to release the unstably bound linkers from thecell-binding agent, and (c) purifying the mixture to provide a purifiedconjugate.

In another embodiment, the process comprises (a) contacting the antibodywith the drug (e.g., DM1 or DM4) to form a mixture comprising theantibody and the drug (e.g., DM1 or DM4); and then contacting themixture comprising the antibody and the drug (e.g., DM1 or DM4) with thecross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1),in a solution having a pH of about 4 to about 9 to provide a mixturecomprising (i) the conjugate, (ii) free drug (e.g., DM1 or DM4), and(iii) reaction by-products, (b) quenching the mixture prepared in step(a) to quench any unreacted drug (e.g., DM1 or DM4) and/or unreactedcross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1),(c) holding the mixture prepared in step (b) to release the unstablybound linkers from the cell-binding agent, and (d) purifying the mixtureto provide a purified conjugate (e.g., Ab-MCC-DM1, Ab-SPDB-DM4 orAb-CX1-1-DM1).

Alternatively, the holding step can be performed after purification ofthe conjugate, followed by an additional purification step.

In a preferred embodiment, the reaction is allowed to proceed tocompletion prior to the holding step. In this regard, the holding stepcan be performed about 1 hour to about 48 hours (e.g., about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours,about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24hours, or about 24 hours to about 48 hours) after the mixture comprisingthe antibody and the drug (e.g., DM1 or DM4) is contacted with thecross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1).

The holding step comprises maintaining the solution at a suitabletemperature (e.g., about 0° C. to about 37° C.) for a suitable period oftime (e.g., about 1 hour to about 1 week, about 1 hour to about 24hours, about 1 hour to about 8 hours, or about 1 hour to about 4 hours)to release the unstably bound linkers from the antibody while notsubstantially releasing the stably bound linkers from the antibody. Inone embodiment, the holding step comprises maintaining the solution atabout 20° C. or less (e.g., about 0° C. to about 18° C., about 4° C. toabout 16° C.), at room temperature (e.g., about 20° C. to about 30° C.or about 20° C. to about 25° C.), or at an elevated temperature (e.g.,about 30° C. to about 37° C.). In one embodiment, the holding stepcomprises maintaining the solution at a temperature of about 16° C. toabout 24° C. (e.g., about 15° C., about 16° C., about 17° C., about 18°C., about 19° C., about 20° C., about 21° C., about 22° C., about 23°C., about 24° C., or about 25° C.). In another embodiment, the holdingstep comprises maintaining the solution at a temperature of about 2° C.to about 8° C. (e.g., about 0° C., about 1° C., about 2° C., about 3°C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C.,about 9° C., or about 10° C.). In another embodiment, the holding stepcomprises maintaining the solution at a temperature of about 37° C.(e.g., about 34° C., about 35° C., about 36° C., about 37° C., about 38°C., about 39° C., or about 40° C.).

The duration of the holding step depends on the temperature and the pHat which the holding step is performed. For example, the duration of theholding step can be substantially reduced by performing the holding stepat elevated temperature, with the maximum temperature limited by thestability of the cell-binding agent-cytotoxic agent conjugate. Theholding step can comprise maintaining the solution for about 1 hour toabout 1 day (e.g., about 1 hour, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about9 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours,about 18 hours, about 20 hours, about 22 hours, or about 24 hours),about 10 hours to about 24 hours, about 12 hours to about 24 hours,about 14 hours to about 24 hours, about 16 hours to about 24 hours,about 18 hours to about 24 hours, about 20 hours to about 24 hours,about 5 hours to about 1 week, about 20 hours to about 1 week, about 12hours to about 1 week (e.g., about 12 hours, about 16 hours, about 20hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5days, about 6 days, or about 7 days), or about 1 day to about 1 week.

In one embodiment, the holding step comprises maintaining the solutionat a temperature of about 2° C. to about 8° C. for a period of at leastabout 12 hours for up to a week. In another embodiment, the holding stepcomprises maintaining the solution at a temperature of about 2° C. toabout 8° C. overnight (e.g., about 12 to about 24 hours, preferablyabout 20 hours).

The pH value for the holding step preferably is about 4 to about 10. Inone embodiment, the pH value for the holding step is about 4 or more,but less than about 6 (e.g., 4 to 5.9) or about 5 or more, but less thanabout 6 (e.g., 5 to 5.9). In another embodiment, the pH values for theholding step range from about 6 to about 10 (e.g., about 6.5 to about 9,about 6 to about 8). For example, pH values for the holding step can beabout 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9,about 9.5, or about 10.

In specific embodiments, the holding step can comprise incubating themixture at 25° C. at a pH of about 6-7.5 for about 12 hours to about 1week, incubating the mixture at 4° C. at a pH of about 4.5-5.9 for about5 hours to about 5 days, or incubating the mixture at 25° C. at a pH ofabout 4.5-5.9 for about 5 hours to about 1 day.

The one-step process may optionally include the addition of sucrose tothe reaction step to increase solubility and recovery of the conjugates.Desirably, sucrose is added at a concentration of about 0.1% (w/v) toabout 20% (w/v) (e.g., about 0.1% (w/v), 1% (w/v), 5% (w/v), 10% (w/v),15% (w/v), or 20% (w/v)). Preferably, sucrose is added at aconcentration of about 1% (w/v) to about 10% (w/v) (e.g., about 0.5%(w/v), about 1% (w/v), about 1.5% (w/v), about 2% (w/v), about 3% (w/v),about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7% (w/v), about 8%(w/v), about 9% (w/v), about 10% (w/v), or about 11% (w/v)). Inaddition, the reaction step also can comprise the addition of abuffering agent. Any suitable buffering agent known in the art can beused. Suitable buffering agents include, for example, a citrate buffer,an acetate buffer, a succinate buffer, and a phosphate buffer. In oneembodiment, the buffering agent is selected from the group consisting ofHEPPSO (N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonicacid)), POPSO (piperazine-1,4-bis-(2-hydroxy-propane-sulfonic acid)dehydrate), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid),HEPPS (EPPS) (4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid), TES(N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), and acombination thereof.

In one embodiment, the one-step process can further comprise the step ofpurifying the mixture to provide purified conjugate (e.g., Ab-MCC-DM1,Ab-SPDB-DM4 or Ab-CX1-1-DM1). Any purification methods known in the artcan be used to purify the conjugates of the present invention. In oneembodiment, the conjugates of the present invention using tangentialflow filtration (TFF), non-adsorptive chromatography, adsorptivechromatography, adsorptive filtration, selective precipitation, or anyother suitable purification process, as well as combinations thereof. Inanother embodiment, prior to subjecting the conjugates to purificationprocess described above, the conjugates are first filtered through oneor more PVDF membranes. Alternatively, the conjugates are filteredthrough one or more PVDF membranes after subjecting the conjugates tothe purification process described above. For example, in oneembodiment, the conjugates are filtered through one or more PVDFmembranes and then purified using tangential flow filtration.Alternatively, the conjugates are purified using tangential flowfiltration and then filtered through one or more PVDF membranes.

Any suitable TFF systems may be utilized for purification, including aPellicon type system (Millipore, Billerica, Mass.), a Sartocon Cassettesystem (Sartorius AG, Edgewood, N.Y.), and a Centrasette type system(Pall Corp., East Hills, N.Y.).

Any suitable adsorptive chromatography resin may be utilized forpurification. Preferred adsorptive chromatography resins includehydroxyapatite chromatography, hydrophobic charge inductionchromatography (HCIC), hydrophobic interaction chromatography (HIC), ionexchange chromatography, mixed mode ion exchange chromatography,immobilized metal affinity chromatography (IMAC), dye ligandchromatography, affinity chromatography, reversed phase chromatography,and combinations thereof. Examples of suitable hydroxyapatite resinsinclude ceramic hydroxyapatite (CHT Type I and Type II, Bio-RadLaboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp.,East Hills, N.Y.), and ceramic fluoroapatite (CFT Type I and Type II,Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable HCICresin is MEP Hypercel resin (Pall Corp., East Hills, N.Y.). Examples ofsuitable HIC resins include Butyl-Sepharose, Hexyl-Sepaharose,Phenyl-Sepharose, and Octyl Sepharose resins (all from GE Healthcare,Piscataway, N.J.), as well as Macro-prep Methyl and Macro-Prep t-Butylresins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ionexchange resins include SP-Sepharose, CM-Sepharose, and Q-Sepharoseresins (all from GE Healthcare, Piscataway, N.J.), and Unosphere S resin(Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable mixedmode ion exchangers include Bakerbond ABx resin (JT Baker, PhillipsburgN.J.). Examples of suitable IMAC resins include Chelating Sepharoseresin (GE Healthcare, Piscataway, N.J.) and Profinity IMAC resin(Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable dyeligand resins include Blue Sepharose resin (GE Healthcare, Piscataway,N.J.) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, Calif.).Examples of suitable affinity resins include Protein A Sepharose resin(e.g., MabSelect, GE Healthcare, Piscataway, N.J.) and lectin affinityresins, e.g. Lentil Lectin Sepharose resin (GE Healthcare, Piscataway,N.J.), where the antibody bears appropriate lectin binding sites.Examples of suitable reversed phase resins include C4, C8, and C18resins (Grace Vydac, Hesperia, Calif.).

Any suitable non-adsorptive chromatography resin may be utilized forpurification. Examples of suitable non-adsorptive chromatography resinsinclude, but are not limited to, SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™resins (e.g., S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 andSUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, andP-100), and others known to those of ordinary skill in the art.

Two-Step Process and One-Pot Process

In one embodiment, the conjugates of the present invention can beprepared as described in the U.S. Pat. No. 7,811,572 and U.S. PatentApplication Publication No. 2006/0182750. The process comprises thesteps of (a) contacting the antibody of the present invention with thecross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB, Sulfo-SPDB or CX1-1)to covalently attach the linker (i.e., Ab-SMCC, Ab-SPDB or Ab-CX1-1) tothe antibody and thereby prepare a first mixture comprising the antibodyhaving the linker bound thereto; (b) optionally subjecting the firstmixture to a purification process to prepare a purified first mixture ofthe antibody having the linker bound thereto; (c) conjugating the drug(e.g., DM1 or DM4) to the antibody having the linker bound thereto inthe first mixture by reacting the antibody having the linker boundthereto with the drug (e.g., DM1 or DM4) in a solution having a pH ofabout 4 to about 9 to prepare a second mixture comprising (i) conjugate(e.g., Ab-MCC-DM1, Ab-SPDB-DM4 or Ab-CX1-1-DM1), (ii) free drug (e.g.,DM1 or DM4); and (iii) reaction by-products; and (d) subjecting thesecond mixture to a purification process to purify the conjugate fromthe other components of the second mixture. Alternatively, thepurification step (b) can be omitted. Any purification methods describedherein can be used for steps (b) and (d). In one embodiment, TFF is usedfor both steps (b) and (d). In another embodiment, TFF is used for step(b) and absorptive chromatography (e.g., CHT) is used for step (d).

One-Step Reagent and In-Situ Process

In one embodiment, the conjugates of the present invention can beprepared by conjugating pre-formed drug-linker compound (e.g., SMCC-DM1,Sulfo-SMCC-DM1, SPDB-DM4 or CX1-1-DM1) to the antibody of the presentinvention, as described in U.S. Pat. No. 6,441,163 and U.S. PatentApplication Publication Nos. 2011/0003969 and 2008/0145374, followed bya purification step. Any purification methods described herein can beused. The drug-linker compound is prepared by reacting the drug (e.g.,DM1 or DM4) with the cross-linking agent (e.g., SMCC, Sulfo-SMCC, SPDB,Sulfo-SPDB or CX1-1). The drug-linker compound (e.g., SMCC-DM1,Sulfo-SMCC-DM1, SPDB-DM4 or CX1-1-DM1) is optionally subjected topurification before being conjugated to the antibody.

4. Characterization and Selection of Desirable Antibodies and AntibodyDrug Conjugates

The antibodies, antibody fragments (e.g., antigen binding fragments) orantibody drug conjugates of the present invention can be characterizedand selected for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

For example, an antibody of the invention can be tested for its antigenbinding activity by known methods such as ELISA, FACS, Biacore orWestern blot.

Transgenic animals and cell lines are particularly useful in screeningantibody drug conjugates (ADCs) that have potential as prophylactic ortherapeutic treatments of cancer overexpression of tumor-associatedantigens and cell surface receptors. Screening for a useful ADC mayinvolve administering a candidate ADC over a range of doses to thetransgenic animal, and assaying at various time points for the effect(s)of the ADC on the disease or disorder being evaluated. Alternatively, oradditionally, the drug can be administered prior to or simultaneouslywith exposure to an inducer of the disease, if applicable. The candidateADC may be screened serially and individually, or in parallel undermedium or high-throughput screening format.

One embodiment is a screening method comprising (a) transplanting cellsfrom a stable cancer cell line or human patient tumor expressing FGFR2or FGFR4 (e.g., a breast cancer cell line or tumor fragment, a gastriccancer cell line or tumor fragment) into a non-human animal, (b)administering an ADC drug candidate to the non-human animal and (c)determining the ability of the candidate to inhibit the growth of tumorsfrom the transplanted cell line. The invention also encompasses a methodof screening ADC candidates for the treatment of a disease or disordercharacterized by the overexpression of FGFR2 or FGFR4 comprising (a)contacting cells from a stable cancer cell line expressing FGFR2 orFGFR4 with a drug candidate, and (b) evaluating the ability of the ADCcandidate to inhibit the growth of the stable cell line.

One embodiment is a screening method comprising (a) contacting cellsfrom a stable cancer cell line expressing FGFR2 or FGFR4 with an ADCdrug candidate and (b) evaluating the ability of the ADC candidate toblock ligand activation of FGFR2 or FGFR4. In another embodiment theability of the ADC candidate to block ligand-stimulated tyrosinephosphorylation is evaluated.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable cancer cell line expressing FGFR2 or FGFR4 with an ADCdrug candidate and (b) evaluating the ability of the ADC candidate toinduce cell death. In one embodiment the ability of the ADC candidate toinduce apoptosis is evaluated.

In one embodiment, candidate ADC are screened by being administered tothe transgenic animal over a range of doses, and evaluating the animal'sphysiological response to the compounds over time. In some cases, it maybe appropriate to administer the compound in conjunction with co-factorsthat would enhance the efficacy of the compound. If cell lines derivedfrom the subject transgenic animals are used to screen for ADCs usefulin treating various disorders associated with overexpression of FGFR2 orFGFR4, the test ADCs are added to the cell culture medium at anappropriate time, and the cellular response to the ADCs is evaluatedover time using the appropriate biochemical and/or histological assays.

Thus, the present invention provides assays for identifying ADC whichspecifically target and bind to both FGFR2 and FGFR4, the overexpressionand amplification of which on the tumor cells is correlated withabnormal cellular function. According to the present invention,anti-FGFR2/4 antibodies or antibody fragments (e.g., antigen bindingfragments) with the following properties are better candidates formaking ADCs: affinity to human FGFR2 of <10 nM, following conjugation toMCC-DM1 or SPDB-DM4, ability to inhibit growth of FGFR2/4 amplifiedand/or overexpressing cells with an IC50 of <2 nM, a slow clearancerate, for example, <45 ml/d/kg in a mouse following a single 3 mg/kg IVdose

FGFR2/4 Antibodies

The present invention provides antibodies or antibody fragments (e.g.,antigen binding fragments) that specifically bind to human FGFR2 andFGFR4 (FGFR2/4). Antibodies or antibody fragments (e.g., antigen bindingfragments) of the invention include, but are not limited to, the humanmonoclonal antibodies or fragments thereof, isolated as described, inthe Examples (see Section 6 below).

The present invention in certain embodiments provides antibodies orantibody fragments (e.g., antigen binding fragments) that specificallybind FGFR2/4, said antibodies or antibody fragments (e.g., antigenbinding fragments) comprise a VH domain having an amino acid sequence ofSEQ ID NO: 7, 27, 47, 67, 87, or 107. The present invention in certainembodiments also provides antibodies or antibody fragments (e.g.,antigen binding fragments) that specifically bind to FGFR2/4, saidantibodies or antibody fragments (e.g., antigen binding fragments)comprise a VH CDR having an amino acid sequence of any one of the VHCDRs listed in Table 1, infra. In particular embodiments, the inventionprovides antibodies or antibody fragments (e.g., antigen bindingfragments) that specifically bind to FGFR2/4, said antibodies comprising(or alternatively, consist of) one, two, three, four, five or more VHCDRs having an amino acid sequence of any of the VH CDRs listed in Table1, infra.

The present invention provides antibodies or antibody fragments (e.g.,antigen binding fragments) that specifically bind to FGFR2/4, saidantibodies or antibody fragments (e.g., antigen binding fragments)comprise a VL domain having an amino acid sequence of SEQ ID NO: 17, 37,57, 77, 97, or 117. The present invention also provides antibodies orantibody fragments (e.g., antigen binding fragments) that specificallybind to FGFR2/4, said antibodies or antibody fragments (e.g., antigenbinding fragments) comprise a VL CDR having an amino acid sequence ofany one of the VL CDRs listed in Table 1, infra. In particular, theinvention provides antibodies or antibody fragments (e.g., antigenbinding fragments) that specifically bind to FGFR2/4, said antibodies orantibody fragments (e.g., antigen binding fragments) comprise (oralternatively, consist of) one, two, three or more VL CDRs having anamino acid sequence of any of the VL CDRs listed in Table 1, infra.

Other antibodies or antibody fragments (e.g., antigen binding fragments)of the invention include amino acids that have been mutated, yet have atleast 60, 70, 80, 90 or 95 percent identity in the CDR regions with theCDR regions depicted in the sequences described in Table 1. In someembodiments, it includes mutant amino acid sequences wherein no morethan 1, 2, 3, 4 or 5 amino acids have been mutated in the CDR regionswhen compared with the CDR regions depicted in the sequence described inTable 1.

The present invention also provides nucleic acid sequences that encodeVH, VL, the full length heavy chain, and the full length light chain ofthe antibodies that specifically bind to FGFR2/4. Such nucleic acidsequences can be optimized for expression in mammalian cells.

TABLE 1 Examples of anti-FGFR24 Antibodies of the Present InventionmAb 12425 SEQ ID NO: 1 HCDR1 (Kabat) DYAMS SEQ ID NO: 2 HCDR2 (Kabat)VIEGDGSYTHYADSVKG SEQ ID NO: 3 HCDR3 (Kabat) EKTYSSAFDY SEQ ID NO: 4HCDR1 (Chothia) GFTFSDY SEQ ID NO: 5 HCDR2 (Chothia) EGDGSY SEQ ID NO: 6HCDR3 (Chothia) EKTYSSAFDY SEQ ID NO: 7 VHQVQLLESGGGLVQPGGSLRLSCAASGETESDYAMSWVRQAPGKGLEWVSVIEGDGSYTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKTYSSAFDYWGQGTLVTVSS SEQ ID NO: 8 DNA VHCAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTGGCGGTAGCCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACCTTTAGCGACTACGCTATGAGCTGGGTCCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGTCAGTGATCGAGGGCGACGGTAGCTACACTCACTACGCCGATAGCGTGAAGGGCCGGTTCACTATCTCTAGGGACAACTCTAAGAACACCCTGTACCTGCAGATGAACTCACTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAAAAGACCTACTCTAGCGCCTTCGACTACTGGGGCCAGGGC ACCCTGGTCACCGTGTCATCASEQ ID NO: 9 Heavy Chain QVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVSVIEGDGSYTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKTYSSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVIVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDILMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 10 DNA Heavy Chain CAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTGGCGGTAGCCTGAGACTGAGCTGTGCCGCCTCCGGCTTCACCTTTAGCGACTACGCTATGAGCTGGGTCCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGTCAGTGATCGAGGGCGACGGTAGCTACACTCACTACGCCGATAGCGTGAAGGGCCGGTTCACTATCTCTAGGGACAACTCTAAGAACACCCTGTACCTGCAGATGAACTCACTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAAAAGACCTACTCTAGCGCCTTCGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTCATCAGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGC AAG SEQ ID NO: 11 (Kabat)LCDR1 RASQDISSDLN SEQ ID NO: 12 (Kabat) LCDR2 DASNLQSSEQ ID NO: 13 (Kabat) LCDR3 QQHYSPSHT SEQ ID NO: 14 (Chothia) LCDR1SQDISSD SEQ ID NO: 15 (Chothia) LCDR2 DAS SEQ ID NO: 16 (Chothia) LCDR3HYSPSH SEQ ID NO: 17 VL DIQMTQSPSSLSASVGDRVTITCRASQDISSDLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFILTISSLQPEDFA VYYCQQHYSPSHTFGQGTKVEIKSEQ ID NO: 18 DNA VL GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCCTCAGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGGACATCTCTAGCGACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCCTCTAACCTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGTAGCGGCTCAGGCACCGACTTTACCCTGACTATCTCTAGCCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTCAGCAGCACTATAGCCCTAGTCACACCTTC GGCCAGGGCACTAAGGTCGAGATTAAGSEQ ID NO: 19 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDISSDLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFILTISSLQPEDFAVYYCQQHYSPSHTFGQGTKVEIKRTVAAPSVFIEPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSEN RGEC SEQ ID NO 20:DNA Light Chain GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCCTCAGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGGACATCTCTAGCGACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACGCCTCTAACCTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGTAGCGGCTCAGGCACCGACTTTACCCTGACTATCTCTAGCCTGCAGCCCGAGGACTTCGCCGTCTACTACTGTCAGCAGCACTATAGCCCTAGTCACACCTTCGGCCAGGGCACTAAGGTCGAGATTAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC mAb 14629SEQ ID NO: 21 HCDR1 (Kabat) SYAIS SEQ ID NO: 22 HCDR2 (Kabat)YISPYMGETHYAQRFQG SEQ ID NO: 23 HCDR3 (Kabat) ESYEYFDI SEQ ID NO: 24HCDR1 (Chothia) GGTFSSY SEQ ID NO: 25 HCDR2 (Chothia) SPYMGESEQ ID NO: 26 HCDR3 (Chothia) ESYEYFDI SEQ ID NO: 27 VHQVQLVQSGAEVKKPGSSVKVSCKASGGIFSSYAISWVRQAPGQGLEWMGYISPYMGETHYAQRFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESYEYFDIWGQGTLVTVSS SEQ ID NO: 28 DNA VHCAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAAACCCGGCTCTAGCGTGAAGGTGTCCTGTAAAGCCTCCGGCGGCACCTTCTCTAGCTACGCTATTAGCTGGGTCCGACAGGCCCCAGGACAGGGCCTGGAATGGATGGGCTATATTAGCCCCTATATGGGCGAGACTCACTACGCTCAGCGGTTTCAGGGTAGAGTGACTATCACCGCCGACGAGTCTACTAGCACCGCCTATATGGAACTGTCTAGCCTGAGATCAGAGGACACCGCCGTCTACTACTGCGCTAGAGAGTCCTACGAGTACTTCGATATCTGGGGCCAGGGCACCCTG GTCACCGTGTCATCA SEQ ID NO: 29Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGGIFSSYAISWVRQAPGQGLEWMGYISPYMGETHYAQRFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESYEYFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDILMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 30 DNA Heavy Chain CAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAAACCCGGCTCTAGCGTGAAGGTGTCCTGTAAAGCCTCCGGCGGCACCTTCTCTAGCTACGCTATTAGCTGGGTCCGACAGGCCCCAGGACAGGGCCTGGAATGGATGGGCTATATTAGCCCCTATATGGGCGAGACTCACTACGCTCAGCGGTTTCAGGGTAGAGTGACTATCACCGCCGACGAGTCTACTAGCACCGCCTATATGGAACTGTCTAGCCTGAGATCAGAGGACACCGCCGTCTACTACTGCGCTAGAGAGTCCTACGAGTACTTCGATATCTGGGGCCAGGGCACCCTGGTCACCGTGTCATCAGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAG SEQ ID NO: 31 (Kabat) LCDR1RASQSISNDLA SEQ ID NO: 32 (Kabat) LCDR2 ATSILQS SEQ ID NO: 33 (Kabat)LCDR3 LQYYDYSYT SEQ ID NO: 34 (Chothia) LCDR1 SQSISNDSEQ ID NO: 35 (Chothia) LCDR2 ATS SEQ ID NO: 36 (Chothia) LCDR3 YYDYSYSEQ ID NO: 37 VL DIQMTQSPSSLSASVGDRVTITCRASQSISNDLAWYQQKPGKAPKLLIYATSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQYYDYSYTFGQGTKVEIKSEQ ID NO: 38 DNA VL GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCCTCAGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCCCAGTCTATCTCTAACGACCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCTACCTCTATCCTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGTAGCGGCTCAGGCACCGACTTTACCCTGACTATCTCTAGCCTGCAGCCCGAGGACTTCGCTACCTACTACTGCCTGCAGTACTACGACTACTCCTACACCTTC GGCCAGGGCACTAAGGTCGAGATTAAGSEQ ID NO: 39 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQSISNDLAWYQQKPGKAPKLLIYATSILQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQYYDYSYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSEN RGEC SEQ ID NO: 40DNA Light Chain GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCCTCAGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCCCAGTCTATCTCTAACGACCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGCTACCTCTATCCTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGTAGCGGCTCAGGCACCGACTTTACCCTGACTATCTCTAGCCTGCAGCCCGAGGACTTCGCTACCTACTACTGCCTGCAGTACTACGACTACTCCTACACCTTCGGCCAGGGCACTAAGGTCGAGATTAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC mAb 20562SEQ ID NO: 41 HCDR1 (Kabat) DYAMS SEQ ID NO: 42 HCDR2 (Kabat)VIEGDASYTHYADSVRG SEQ ID NO: 43 HCDR3 (Kabat) ERTYSSAFDY SEQ ID NO: 44HCDR1 (Chothia) GFTFSDY SEQ ID NO: 45 HCDR2 (Chothia) EGDASYSEQ ID NO: 46 HCDR3 (Chothia) ERTYSSAFDY SEQ ID NO: 47 VHEVQLLESGGGLVQPGGSLRLSCAASGFTESDYAMSWVRQAPGKGLEWVSVIEGDASYTHYADSVRGRETISRDNSKNTLYLQMNSLRAEDTAVYYCARERTYSSAFDYWGQGTLVTVSS SEQ ID NO: 48 DNA VHGAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTTAGCGACTACGCTATGAGCTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCAGCGTGATCGAGGGCGACGCTAGTTACACTCACTACGCCGATAGCGTCAGAGGCCGGTTCACTATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAGCGGACCTACTCTAGCGCCTTCGACTACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGCSEQ ID NO: 49 Heavy Chain EVQLLESGGGLVQPGGSLRLSCAASGFTESDYAMSWVRQAPGKGLEWVSVIEGDASYTHYADSVRGRETISRDNSKNTLYLQMNSLRAEDTAVYYCARERTYSSAFDYWGQGTLVTVSSASTKGPSVEPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDILMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 50 DNA Heavy Chain GAGGTGCAGCTGCTGGAATCAGGCGGCGGACTGGTGCAGCCTGGCGGTAGCCTGAGACTGAGCTGCGCTGCTAGTGGCTTCACCTTTAGCGACTACGCTATGAGCTGGGTCAGACAGGCCCCTGGTAAAGGCCTGGAGTGGGTCAGCGTGATCGAGGGCGACGCTAGTTACACTCACTACGCCGATAGCGTCAGAGGCCGGTTCACTATCTCTAGGGATAACTCTAAGAACACCCTGTACCTGCAGATGAATAGCCTGAGAGCCGAGGACACCGCCGTCTACTACTGCGCTAGAGAGCGGACCTACTCTAGCGCCTTCGACTACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCAAGTGTGTTTCCCCTGGCCCCCAGCAGCAAGTCTACTTCCGGCGGAACTGCTGCCCTGGGTTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCCTGGAACTCTGGGGCTCTGACTTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCTCCAGCTCTCTGGGAACCCAGACCTATATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCTCCAGAACTGCTGGGAGGGCCTTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTGCCAGCCCCAATCGAAAAGACAATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGC AAG SEQ ID NO: 51LCDR1 (Kabat) RASQDISSDLN SEQ ID NO: 52 LCDR2 (Kabat) DASNLQSSEQ ID NO: 53 LCDR3 (Kabat) QQHYSPSHT SEQ ID NO: 54 LCDR1 (Chothia)SQDISSD SEQ ID NO: 55 LCDR2 (Chothia) DAS SEQ ID NO: 56 LCDR3 (Chothia)HYSPSH SEQ ID NO: 57 VL DIQMTQSPSSLSASVGDRVTITCRASQDISSDLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDETLTISSLQPEDFA TYYCQQHYSPSHTFGQGTKVEIKSEQ ID NO: 58 DNA VL GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCTAGTGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGGATATCTCTAGCGACCTGAACTGGTATCAGCAGAAGCCCGGTAAAGCCCCTAAGCTGCTGATCTACGACGCCTCTAACCTGCAGTCAGGCGTGCCCTCTAGGTTTAGCGGTAGCGGTAGTGGCACCGACTTCACCCTGACTATCTCTAGCCTGCAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGCACTATAGCCCTAGTCACACCTTC GGTCAGGGCACTAAGGTCGAGATTAAGSEQ ID NO: 59 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDISSDLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSPSHTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSEN RGEC SEQ ID NO: 60DNA Light Chain GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCTAGTGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGGATATCTCTAGCGACCTGAACTGGTATCAGCAGAAGCCCGGTAAAGCCCCTAAGCTGCTGATCTACGACGCCTCTAACCTGCAGTCAGGCGTGCCCTCTAGGTTTAGCGGTAGCGGTAGTGGCACCGACTTCACCCTGACTATCTCTAGCCTGCAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGCACTATAGCCCTAGTCACACCTTCGGTCAGGGCACTAAGGTCGAGATTAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC mAb 20811SEQ ID NO: 61 HCDR1 (Kabat) DYAMS SEQ ID NO: 62 HCDR2 (Kabat)TIEGDSNYIEYADSVKG SEQ ID NO: 63 HCDR3 (Kabat) ERTYSSAFDY SEQ ID NO: 64HCDR1 (Chothia) GFTFSDY SEQ ID NO: 65 HCDR2 (Chothia) EGDSNYSEQ ID NO: 66 HCDR3 (Chothia) ERTYSSAFDY SEQ ID NO: 67 VHQVQLLESGGGLVQPGGSLRLSCAASGETESDYAMSWVRQAPGKGLEWVSTIEGDSNYIEYADSVKGRETISRDNSKNTLYLQMNSLRAEDTAVYYCARERTYSSAFDYWGQGTLVTVSS SEQ ID NO: 68 DNA VHCAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCACCTTTTCTGACTACGCTATGTCTTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTGGGTTTCCACTATCGAAGGTGACAGCAACTACATCGAATATGCGGATAGCGTGAAAGGCCGCTTTACCATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGAACGTACTTACTCTTCTGCTTTCGATTACTGGGGCCAAGGC ACCCTGGTGACTGTTAGCTCASEQ ID NO: 69 Heavy Chain QVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVSTIEGDSNYIEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERTYSSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDILMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 70 DNA Heavy Chain CAGGTGCAATTGCTGGAAAGCGGCGGTGGCCTGGTGCAGCCGGGTGGCAGCCTGCGTCTGAGCTGCGCGGCGTCCGGATTCACCTTTTCTGACTACGCTATGTCTTGGGTGCGCCAGGCCCCGGGCAAAGGTCTCGAGTGGGTTTCCACTATCGAAGGTGACAGCAACTACATCGAATATGCGGATAGCGTGAAAGGCCGCTTTACCATCAGCCGCGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGAACGTACTTACTCTTCTGCTTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAA SEQ ID NO: 71 (Kabat)LCDR1 RASQDISSDLN SEQ ID NO: 72 (Kabat) LCDR2 DASNLQSSEQ ID NO: 73 (Kabat) LCDR3 HQWYSTLYT SEQ ID NO: 74 (Chothia) LCDR1SQDISSD SEQ ID NO: 75 (Chothia) LCDR2 DAS SEQ ID NO: 76 (Chothia) LCDR3WYSTLY SEQ ID NO: 77 VL DIQMTQSPSSLSASVGDRVTITCRASQDISSDLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCHQWYSTLYTFGQGTKVEIKSEQ ID NO: 78 DNA VL GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGGACATTTCTTCTGACCTGAACTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTAACCTGCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCATCAGTGGTACTCTACTCTGTACACCTTT GGCCAGGGCACGAAAGTTGAAATTAAASEQ ID NO: 79 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDISSDLNWYQQKPGKAPKLLIYDASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQWYSTLYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSEN RGEC SEQ ID NO: 80DNA Light Chain GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGGACATTTCTTCTGACCTGAACTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGACGCTTCTAACCTGCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCATCAGTGGTACTCTACTCTGTACACCTTTGGCCAGGGCACGAAAGTTGAAATTAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGT mAb 12422SEQ ID NO: 81 HCDR1 (Kabat) SYAIS SEQ ID NO: 82 HCDR2 (Kabat)YISPYMGETHYAQKFQG SEQ ID NO: 83 HCDR3 (Kabat) ESYEYFDI SEQ ID NO: 84HCDR1 (Chothia) GGTFSSY SEQ ID NO: 85 HCDR2 (Chothia) SPYMGESEQ ID NO: 86 HCDR3 (Chothia) ESYEYFDI SEQ ID NO: 87 VHQVQLVQSGAEVKKPGSSVKVSCKASGGTESSYAISWVRQAPGQGLEWMGYISPYMGETHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESVEYEDIWGQGTLVTVSS SEQ ID NO: 88 DNA VHCAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCTACATCTCTCCGTACATGGGCGAAACTCATTACGCCCAGAAATTTCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGAATCTTACGAATACTTCGACATCTGGGGCCAAGGCACCCTG GTGACTGTTAGCTCA SEQ ID NO: 89Heavy Chain QVQLVQSGAEVKKPGSSVKVSCKASGGTESSYAISWVRQAPGQGLEWMGYISPYMGETHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARESVEYEDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVELEPPKPKDILMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLICLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYSKLTVDKSRWQQG NVESCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 90 DNA Heavy Chain CAGGTGCAATTGGTGCAGAGCGGTGCCGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTTAGCTGCAAAGCATCCGGAGGGACGTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCTACATCTCTCCGTACATGGGCGAAACTCATTACGCCCAGAAATTTCAGGGCCGGGTGACCATTACCGCCGATGAAAGCACCAGCACCGCCTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGAATCTTACGAATACTTCGACATCTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 91 (Kabat) LCDR1RASQSISNDLA SEQ ID NO: 92 (Kabat) LCDR2 ATSILQS SEQ ID NO: 93 (Kabat)LCDR3 LQYYDYSYT SEQ ID NO: 94 (Chothia) LCDR1 SQSISNDSEQ ID NO: 95 (Chothia) LCDR2 ATS SEQ ID NO: 96 (Chothia) LCDR3 YYDYSYSEQ ID NO: 97 VL DIQMTQSPSSLSASVGDRVTITCRASQSISNDLAWYQQKPGKAPKLLIYATSILQSGVPSRFSGSGSGTDFILTISSLOPEDFA TYYCLQYYDYSYTEGQGTKVEIKSEQ ID NO: 98 DNA VL GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGTCTATTTCTAACGACCTGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGCTACTTCTATCCTGCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCTGCAGTACTACGACTACTCTTACACCTTT GGCCAGGGCACGAAAGTTGAAATTAAASEQ ID NO: 99 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQSISNDLAWYQQKPGKAPKLLIYATSILQSGVPSRFSGSGSGTDFILTISSLOPEDFATYYCLQYYDYSYTEGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSEN RGEC SEQ ID NO: 100DNA Light Chain GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGTCTATTTCTAACGACCTGGCTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGCTACTTCTATCCTGCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCTGCAGTACTACGACTACTCTTACACCTTTGGCCAGGGCACGAAAGTTGAAATTAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGT mAb 12439SEQ ID NO: 101 HCDR1 (Kabat) SYDIS SEQ ID NO: 102 HCDR2 (Kabat)WINPYNGGTNYAQKFQG SEQ ID NO: 103 HCDR3 (Kabat) EGSGMIVYPGWSYAFDYSEQ ID NO: 104 HCDR1 (Chothia) GYTFTSY SEQ ID NO: 105 HCDR2 (Chothia)NPYNGG SEQ ID NO: 106 HCDR3 (Chothia) EGSGMIVYPGWSYAFDY SEQ ID NO: 107VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDISWVRQAPGQGLEWMGWINPYNGGINYAQKFOGRVIMTRDTSISTAYMELSRLRSEDTAVYYCAREGSGMIVYPGW5YAFDYWGQGTLVTVSS SEQ ID NO: 108 DNA VHCAGGTGCAATTGGTGCAGAGCGGTGCGGAAGTGAAAAAACCGGGTGCCAGCGTGAAAGTTAGCTGCAAAGCGTCCGGATATACCTTCACTTCTTACGACATCTCTTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCTGGATCAACCCGTACAACGGCGGTACGAACTACGCGCAGAAATTTCAGGGCCGGGTGACCATGACCCGTGATACCAGCATTAGCACCGCGTATATGGAACTGAGCCGTCTGCGTAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGAAGGTTCTGGTATGATCGTTTACCCGGGTTGGTCTTACGCTTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCA SEQ ID NO: 109 Heavy ChainQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDISWVRQAPGQGLEWMGWINPYNGGINYAQKFOGRVIMTRDTSISTAYMELSRLRSEDTAVYYCAREGSGMIVYPGW5YAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS5GLYSISSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDILMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLICLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 110 DNA Heavy ChainCAGGTGCAATTGGTGCAGAGCGGTGCGGAAGTGAAAAAACCGGGTGCCAGCGTGAAAGTTAGCTGCAAAGCGTCCGGATATACCTTCACTTCTTACGACATCTCTTGGGTGCGCCAGGCCCCGGGCCAGGGCCTCGAGTGGATGGGCTGGATCAACCCGTACAACGGCGGTACGAACTACGCGCAGAAATTTCAGGGCCGGGTGACCATGACCCGTGATACCAGCATTAGCACCGCGTATATGGAACTGAGCCGTCTGCGTAGCGAAGATACGGCCGTGTATTATTGCGCGCGTGAAGGTTCTGGTATGATCGTTTACCCGGGTTGGTCTTACGCTTTCGATTACTGGGGCCAAGGCACCCTGGTGACTGTTAGCTCAGCCTCCACCAAGGGTCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAASEQ ID NO: 111 (Kabat) LCDR1 RASQDISNDLG SEQ ID NO: 112 (Kabat) LCDR2AASSLQS SEQ ID NO: 113 (Kabat) LCDR3 QQHYHTPNT SEQ ID NO: 114 (Chothia)LCDR1 SQDISND SEQ ID NO: 115 (Chothia) LCDR2 AASSEQ ID NO: 116 (Chothia) LCDR3 HYHTPN SEQ ID NO: 117 VLDIQMTQSPSSLSASVGDRVTITCRASQDISNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFILTISSLOPEDFA TYYCQQHYHTPNTFGQGTKVEIKSEQ ID NO: 118 DNA VL GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGGACATTTCTAACGACCTGGGTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGCTGCTTCTTCTCTGCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCAGCAGCATTACCATACTCCGAACACCTTT GGCCAGGGCACGAAAGTTGAAATTAAASEQ ID NO: 119 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDISNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFILTISSLOPEDFATYYCQQHYHTPNTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVIKSEN RGEC SEQ ID NO: 120DNA Light Chain GATATCCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCCAGCGTGGGCGATCGCGTGACCATTACCTGCAGAGCCAGCCAGGACATTTCTAACGACCTGGGTTGGTACCAGCAGAAACCGGGCAAAGCGCCGAAACTATTAATCTACGCTGCTTCTTCTCTGCAAAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATTAGCTCTCTGCAACCGGAAGACTTTGCGACCTATTATTGCCAGCAGCATTACCATACTCCGAACACCTTTGGCCAGGGCACGAAAGTTGAAATTAAACGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC CGGGGCGAGTGT

Other antibodies of the invention include those where the amino acids ornucleic acids encoding the amino acids have been mutated, yet have atleast 60, 70, 80, 90 or 95 percent identity to the sequences describedin Table 1. In some embodiments, it include mutant amino acid sequenceswherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated inthe variable regions when compared with the variable regions depicted inthe sequence described in Table 1, while retaining substantially thesame therapeutic activity.

Since each of these antibodies can bind to FGFR2/4, the VH, VL, fulllength light chain, and full length heavy chain sequences (amino acidsequences and the nucleotide sequences encoding the amino acidsequences) can be “mixed and matched” to create other FGFR2/4-bindingantibodies of the invention. Such “mixed and matched” FGFR2/4-bindingantibodies can be tested using the binding assays known in the art(e.g., ELISAs, and other assays described in the Example section). Whenthese chains are mixed and matched, a VH sequence from a particularVH/VL pairing should be replaced with a structurally similar VHsequence. Likewise a full length heavy chain sequence from a particularfull length heavy chain/full length light chain pairing should bereplaced with a structurally similar full length heavy chain sequence.Likewise, a VL sequence from a particular VH/VL pairing should bereplaced with a structurally similar VL sequence. Likewise a full lengthlight chain sequence from a particular full length heavy chain/fulllength light chain pairing should be replaced with a structurallysimilar full length light chain sequence. Accordingly, in one aspect,the invention provides an isolated monoclonal antibody or antigenbinding region thereof having: a heavy chain variable region comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:7, 27, 47, 67, 87 and 107; and a light chain variable region comprisingan amino acid sequence selected from the group consisting of SEQ ID NOs:17, 37, 57, 77, 97, and 117; wherein the antibody specifically binds toFGFR2/4.

In another aspect, the invention provides (i) an isolated monoclonalantibody having: a full length heavy chain comprising an amino acidsequence that has been optimized for expression in the cell of amammalian selected from the group consisting of SEQ ID NOs: 9, 29, 49,69, 89, and 109; and a full length light chain comprising an amino acidsequence that has been optimized for expression in the cell of amammalian selected from the group consisting of SEQ ID NOs: 19, 39, 59,79, 99, and 119; or (ii) a functional protein comprising an antigenbinding portion thereof.

In another aspect, the present invention provides FGFR2/4-bindingantibodies that comprise the heavy chain and light chain CDR1s, CDR2sand CDR3s as described in Table 1, or combinations thereof. The aminoacid sequences of the VH CDR1s of the antibodies are shown in SEQ IDNOs: 1, 21, 41, 61, 81, and 101. The amino acid sequences of the VHCDR2s of the antibodies and are shown in SEQ ID NOs: 2, 22, 42, 62, 82,and 102. The amino acid sequences of the VH CDR3s of the antibodies areshown in SEQ ID NOs: 3, 23, 43, 63, 83, and 103. The amino acidsequences of the VL CDR1s of the antibodies are shown in SEQ ID NOs: 11,31, 51, 71, 91, and 111. The amino acid sequences of the VL CDR2s of theantibodies are shown in SEQ ID NOs 12, 32, 52, 72, 92, and 112. Theamino acid sequences of the VL CDR3s of the antibodies are shown in SEQID NOs: 13, 33, 53, 73, 93, and 113.

Given that each of these antibodies can bind to FGFR2/4 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequencescan be “mixed and matched” (i.e., CDRs from different antibodies can bemixed and match, although each antibody must contain a VH CDR1, 2 and 3and a VL CDR1, 2 and 3 to create other C5-binding binding molecules ofthe invention. Such “mixed and matched” FGFR2/4-binding antibodies canbe tested using the binding assays known in the art and those describedin the Examples (e.g., ELISAs). When VH CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular VHsequence should be replaced with a structurally similar CDR sequence(s).Likewise, when VL CDR sequences are mixed and matched, the CDR1, CDR2and/or CDR3 sequence from a particular VL sequence should be replacedwith a structurally similar CDR sequence(s). It will be readily apparentto the ordinarily skilled artisan that novel VH and VL sequences can becreated by substituting one or more VH and/or VL CDR region sequenceswith structurally similar sequences from the CDR sequences shown hereinfor monoclonal antibodies of the present invention.

Accordingly, the present invention provides an isolated monoclonalantibody or antigen binding region thereof comprising a heavy chain CDR1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1, 21, 41, 61, 81, and 101; a heavy chain CDR2 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 2,22, 42, 62, 82, and 102; a heavy chain CDR3 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 3, 23, 43,63, 83, and 103; a light chain CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 11, 31, 51, 71, 91,and 111; a light chain CDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 12, 32, 52, 72, 92, and 112;and a light chain CDR3 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 13, 33, 53, 73, 93, and 113; whereinthe antibody specifically binds FGFR2/4.

In a specific embodiment, an antibody or antibody fragment (e.g.,antigen binding fragments) that specifically binds to FGFR2/4 comprisinga heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2;a heavy chain CDR3 of SEQ ID NO:3; a light chain CDR1 of SEQ ID NO: 11;a light chain CDR2 of SEQ ID NO: 12; and a light chain CDR3 of SEQ IDNO: 13.

In a specific embodiment, an antibody or antibody fragment (e.g.,antigen binding fragments) that specifically binds to FGFR2/4 comprisinga heavy chain CDR1 of SEQ ID NO:21, a heavy chain CDR2 of SEQ ID NO: 22;a heavy chain CDR3 of SEQ ID NO:23; a light chain CDR1 of SEQ ID NO:31;a light chain CDR2 of SEQ ID NO: 32; and a light chain CDR3 of SEQ IDNO: 33.

In a specific embodiment, an antibody or antibody fragment (e.g.,antigen binding fragments) that specifically binds to FGFR2/4 comprisinga heavy chain CDR1 of SEQ ID NO:41, a heavy chain CDR2 of SEQ ID NO: 42;a heavy chain CDR3 of SEQ ID NO:43; a light chain CDR1 of SEQ ID NO:51;a light chain CDR2 of SEQ ID NO: 52; and a light chain CDR3 of SEQ IDNO: 53.

In a specific embodiment, an antibody or antibody fragment (e.g.,antigen binding fragments) that specifically binds to FGFR2/4 comprisinga heavy chain CDR1 of SEQ ID NO:61, a heavy chain CDR2 of SEQ ID NO: 62;a heavy chain CDR3 of SEQ ID NO:63; a light chain CDR1 of SEQ ID NO:71;a light chain CDR2 of SEQ ID NO: 72; and a light chain CDR3 of SEQ IDNO: 73.

In a specific embodiment, an antibody or antibody fragment (e.g.,antigen binding fragments) that specifically binds to FGFR2/4 comprisinga heavy chain CDR1 of SEQ ID NO:81, a heavy chain CDR2 of SEQ ID NO: 82;a heavy chain CDR3 of SEQ ID NO:83; a light chain CDR1 of SEQ ID NO:91;a light chain CDR2 of SEQ ID NO: 92; and a light chain CDR3 of SEQ IDNO: 93.

In a specific embodiment, an antibody or antibody fragment (e.g.,antigen binding fragments) that specifically binds to FGFR2/4 comprisinga heavy chain CDR1 of SEQ ID NO: 101, a heavy chain CDR2 of SEQ ID NO:102; a heavy chain CDR3 of SEQ ID NO: 103; a light chain CDR1 of SEQ IDNO: 111; a light chain CDR2 of SEQ ID NO: 112; and a light chain CDR3 ofSEQ ID NO: 113.

In certain embodiments, an antibody that specifically binds to FGFR2/4is an antibody or antibody fragment (e.g., antigen binding fragment)that is described in Table 1.

1. Identification of Epitopes and Antibodies that Bind to the SameEpitope

The present invention provides antibodies and antibody fragments (e.g.,antigen binding fragments) that bind to an epitope of 160-189(KMEKRLHAVPAANTVKFRCPAGGNPMPTMR; SEQ ID NO: 136) and 198-216(KMEKRLHAVPAANTVKFRC; SEQ ID NO: 141) amino acids (numbered according toP21802-3) of human FGFR2.

In some aspects, the present invention provides antibodies or antibodyfragments (e.g., antigen binding fragments) that recognize amino acidsat positions 173 (Asn), 174 (Thr), 175 (Val), 176 (Lys), 178 (Arg), 208(Lys), 209 (Val), 210 (Arg), 212 (Gln), 213 (His), 217 (Ile), 219 (Glue)of human FGFR2 as shown in SEQ ID NO: 137. In another embodiment, thepresent invention provides antibodies or antibody fragments (e.g.,antigen binding fragments) that recognize amino acids at least inpositions 176 (Lys) and 210 (Arg) of human FGFR2 as shown in SEQ ID NO:137.

The present invention also provides antibodies and antibody fragments(e.g., antigen binding fragments) that bind to the same epitope as dothe anti-FGFR2/4 antibodies described in Table 1. Additional antibodiesand antibody fragments (e.g., antigen binding fragments) can thereforebe identified based on their ability to cross-compete (e.g., tocompetitively inhibit the binding of, in a statistically significantmanner) with other antibodies of the invention in FGFR2 and/or FGFR4binding assays. The ability of a test antibody to inhibit the binding ofantibodies and antibody fragments (e.g., antigen binding fragments) ofthe present invention to a FGFR2 and/or FGFR4 protein (e.g., human FGFR2and/or FGFR4) demonstrates that the test antibody can compete with thatantibody or antibody fragment (e.g., antigen binding fragments) forbinding to FGFR2 and/or FGFR4; such an antibody may, according tonon-limiting theory, bind to the same or a related (e.g., a structurallysimilar or spatially proximal) epitope on the FGFR2 and/or FGFR4 proteinas the antibody or antibody fragment (e.g., antigen binding fragments)with which it competes. In a certain embodiment, the antibody that bindsto the same epitope on FGFR2 and/or FGFR4 as the antibodies or antibodyfragments (e.g., antigen binding fragments) of the present invention isa human or humanized monoclonal antibody. Such human or humanizedmonoclonal antibodies can be prepared and isolated as described herein.

2. Further Alteration of the Framework of Fc Region

The present invention provides site-specific labeled immunoconjugates.The immunoconjugates of the invention may comprise modified antibodiesor antigen binding fragments thereof that further comprise modificationsto framework residues within VH and/or VL, e.g. to improve theproperties of the antibody. Typically such framework modifications aremade to decrease the immunogenicity of the antibody. For example, oneapproach is to “back-mutate” one or more framework residues to thecorresponding germline sequence. More specifically, an antibody that hasundergone somatic mutation may contain framework residues that differfrom the germline sequence from which the antibody is derived. Suchresidues can be identified by comparing the antibody framework sequencesto the germline sequences from which the antibody is derived. To returnthe framework region sequences to their germline configuration, thesomatic mutations can be “back-mutated” to the germline sequence by, forexample, site-directed mutagenesis. Such “back-mutated” antibodies arealso intended to be encompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T-cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in, e.g., U.S. Pat.Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described in,e.g., U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described in, e.g., the PCT Publication WO 94/29351 byBodmer et al. In a specific embodiment, one or more amino acids of anantibody or antigen binding fragment thereof of the present inventionare replaced by one or more allotypic amino acid residues, such as thoseshown in FIG. 4 for the IgG1 subclass and the kappa isotype. Allotypicamino acid residues also include, but are not limited to, the constantregion of the heavy chain of the IgG1, IgG2, and IgG3 subclasses as wellas the constant region of the light chain of the kappa isotype asdescribed by Jefferis et al., MAbs. 1:332-338 (2009).

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed in, e.g., the PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al., J. Biol. Chem. 276:6591-6604, 2001).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen.” Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lecl3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., Nat. Biotech. 17:176-180, 1999).

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half-life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

3. Production of the FGFR2/4 Antibodies

Anti-FGFR2/4 antibodies and antibody fragments (e.g., antigen bindingfragments) thereof can be produced by any means known in the art,including but not limited to, recombinant expression, chemicalsynthesis, and enzymatic digestion of antibody tetramers, whereasfull-length monoclonal antibodies can be obtained by, e.g., hybridoma orrecombinant production. Recombinant expression can be from anyappropriate host cells known in the art, for example, mammalian hostcells, bacterial host cells, yeast host cells, insect host cells, etc.

The invention further provides polynucleotides encoding the antibodiesdescribed herein, e.g., polynucleotides encoding heavy or light chainvariable regions or segments comprising the complementarity determiningregions as described herein. In some embodiments, the polynucleotideencoding the heavy chain variable regions has at least 85%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acidsequence identity with a polynucleotide selected from the groupconsisting of SEQ ID NOs: 8, 28, 48, 68, 88, and 108. In someembodiments, the polynucleotide encoding the light chain variableregions has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% nucleic acid sequence identity with a polynucleotideselected from the group consisting of SEQ ID NOs: 18, 38, 58, 78, 98,and 118.

In some embodiments, the polynucleotide encoding the heavy chain has atleast 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% nucleic acid sequence identity with a polynucleotide of SEQ ID NO:10, 30, 50, 70, 90, or 110. In some embodiments, the polynucleotideencoding the light chain has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with apolynucleotide of SEQ ID NO: 20, 40, 60, 80, 100, or 120.

The polynucleotides of the invention can encode only the variable regionsequence of an anti-FGFR2/4 antibody. They can also encode both avariable region and a constant region of the antibody. Some of thepolynucleotide sequences encode a polypeptide that comprises variableregions of both the heavy chain and the light chain of one of theexemplified mouse anti-FGFR2 and/or FGFR4 antibody. Some otherpolynucleotides encode two polypeptide segments that respectively aresubstantially identical to the variable regions of the heavy chain andthe light chain of one of the mouse antibodies.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding an anti-FGFR2/4 antibody orits binding fragment. Direct chemical synthesis of nucleic acids can beaccomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications,Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila etal., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the invention are expression vectors and host cells forproducing the anti-FGFR2/4 antibodies described above. Variousexpression vectors can be employed to express the polynucleotidesencoding the anti-FGFR2/4 antibody chains or binding fragments. Bothviral-based and nonviral expression vectors can be used to produce theantibodies in a mammalian host cell. Nonviral vectors and systemsinclude plasmids, episomal vectors, typically with an expressioncassette for expressing a protein or RNA, and human artificialchromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997). Forexample, nonviral vectors useful for expression of the anti-FGFR2/4polynucleotides and polypeptides in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors includevectors based on retroviruses, adenoviruses, adenoassociated viruses,herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barrvirus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brentet al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeldet al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding an anti-FGFR2/4antibody chain or fragment. In some embodiments, an inducible promoteris employed to prevent expression of inserted sequences except underinducing conditions. Inducible promoters include, e.g., arabinose, lacZ,metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of an anti-FGFR2/4 antibody chain or fragment.These elements typically include an ATG initiation codon and adjacentribosome binding site or other sequences. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf et al., Results Probl. CellDiffer. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516,1987). For example, the SV40 enhancer or CMV enhancer may be used toincrease expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedanti-FGFR2/4 antibody sequences. More often, the inserted anti-FGFR2/4antibody sequences are linked to a signal sequences before inclusion inthe vector. Vectors to be used to receive sequences encodinganti-FGFR2/4 antibody light and heavy chain variable domains sometimesalso encode constant regions or parts thereof. Such vectors allowexpression of the variable regions as fusion proteins with the constantregions thereby leading to production of intact antibodies or fragmentsthereof. Typically, such constant regions are human.

The host cells for harboring and expressing the anti-FGFR2/4 antibodychains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters typicallycontrol expression, optionally with an operator sequence, and haveribosome binding site sequences and the like, for initiating andcompleting transcription and translation. Other microbes, such as yeast,can also be employed to express anti-FGFR2/4 polypeptides of theinvention. Insect cells in combination with baculovirus vectors can alsobe used.

In some preferred embodiments, mammalian host cells are used to expressand produce the anti-FGFR2/4 polypeptides of the present invention. Forexample, they can be either a hybridoma cell line expressing endogenousimmunoglobulin genes (e.g., the myeloma hybridoma clones as described inthe Examples) or a mammalian cell line harboring an exogenous expressionvector (e.g., the SP2/0 myeloma cells exemplified below). These includeany normal mortal or normal or abnormal immortal animal or human cell.For example, a number of suitable host cell lines capable of secretingintact immunoglobulins have been developed, including the CHO celllines, various Cos cell lines, HeLa cells, myeloma cell lines,transformed B-cells and hybridomas. The use of mammalian tissue cellculture to express polypeptides is discussed generally in, e.g.,Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987.Expression vectors for mammalian host cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (see, e.g., Queen et al., Immunol. Rev. 89:49-68, 1986), andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. These expression vectors usually contain promoters derivedfrom mammalian genes or from mammalian viruses. Suitable promoters maybe constitutive, cell type-specific, stage-specific, and/or modulatableor regulatable. Useful promoters include, but are not limited to, themetallothionein promoter, the constitutive adenovirus major latepromoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter,the MRP polIII promoter, the constitutive MPSV promoter, thetetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), the constitutive CMV promoter, and promoter-enhancercombinations known in the art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts (see generallySambrook et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express anti-FGFR2 and/or FGFR4 antibody chains orbinding fragments can be prepared using expression vectors of theinvention which contain viral origins of replication or endogenousexpression elements and a selectable marker gene. Following introductionof the vector, cells may be allowed to grow for 1-2 days in an enrichedmedia before they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype.

Therapeutic and Diagnostic Uses

The antibodies, antibody fragments (e.g., antigen binding fragments),and antibody drug conjugates of the invention are useful in a variety ofapplications including, but not limited to, treatment of cancer, such assolid cancers. In certain embodiments, the antibodies, antibodyfragments (e.g., antigen binding fragments), and antibody drugconjugates of the invention are useful for inhibiting tumor growth,inducing differentiation, reducing tumor volume, and/or reducing thetumorigenicity of a tumor. The methods of use can be in vitro, ex vivo,or in vivo methods.

In one aspect, the antibodies, antibody fragments (e.g., antigen bindingfragments), and antibody drug conjugates of the invention are useful fordetecting the presence of FGFR2 or FGFR4 in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue. In certain embodiments, such tissues include normal and/orcancerous tissues that express FGFR2 or FGFR4 at higher levels relativeto other tissues.

In one aspect, the invention provides a method of detecting the presenceof FGFR2 or FGFR4 in a biological sample. In certain embodiments, themethod comprises contacting the biological sample with an anti-FGFR2/4antibody under conditions permissive for binding of the antibody to theantigen, and detecting whether a complex is formed between the antibodyand the antigen.

In one aspect, the invention provides a method of diagnosing a disorderassociated with increased expression of FGFR2 or FGFR4. In certainembodiments, the method comprises contacting a test cell with ananti-FGFR2/4 antibody; determining the level of expression (eitherquantitatively or qualitatively) of FGFR2 or FGFR4 on the test cell bydetecting binding of the anti-FGFR2/4 antibody to the FGFR2 or FGFR4antigen; and comparing the level of expression of FGFR2 or FGFR4 ib thetest cell with the level of expression of FGFR2 or FGFR4 on a controlcell (e.g., a normal cell of the same tissue origin as the test cell ora cell that expresses FGFR2 or FGFR4 at levels comparable to such anormal cell), wherein a higher level of expression of FGFR2 or FGFR4 onthe test cell as compared to the control cell indicates the presence ofa disorder associated with increased expression of FGFR2 or FGFR4. Incertain embodiments, the test cell is obtained from an individualsuspected of having a disorder associated with increased expression ofFGFR2 or FGFR4. In certain embodiments, the disorder is a cellproliferative disorder, such as a cancer or a tumor.

In certain embodiments, a method of diagnosis or detection, such asthose described above, comprises detecting binding of an anti-FGFR24antibody to FGFR2 or FGFR4 expressed on the surface of a cell or in amembrane preparation obtained from a cell expressing FGFR2 or FGFR4 onits surface. An exemplary assay for detecting binding of an anti-FGFR2/4antibody to FGFR2 or FGFR4 expressed on the surface of a cell is a“FACS” assay.

Certain other methods can be used to detect binding of anti-FGFR2/4antibodies to FGFR2 or FGFR4. Such methods include, but are not limitedto, antigen-binding assays that are well known in the art, such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, fluorescentimmunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-FGFR2/4 antibodies are labeled. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction.

In certain embodiments, anti-FGFR2/4 antibodies are immobilized on aninsoluble matrix. Immobilization entails separating the anti-FGFR2/4antibody from any FGFR2 or FGFR4 proteins that remains free in solution.This conventionally is accomplished by either insolubilizing theanti-FGFR2/4 antibody before the assay procedure, as by adsorption to awater-insoluble matrix or surface (Bennich et al, U.S. Pat. No.3,720,760), or by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-FGFR2/4 antibody afterformation of a complex between the anti-FGFR2/4 antibody and FGFR2 orFGFR4 protein, e.g., by immunoprecipitation.

Any of the above embodiments of diagnosis or detection can be carriedout using an immunoconjugate of the invention in place of or in additionto an anti-FGFR2/4 antibody.

In one embodiment, the invention provides a method of treating,preventing or ameliorating a disease comprising administering theantibodies, antibody fragments (e.g., antigen binding fragments), andantibody drug conjugates of the invention to a patient, thereby treatingthe disease. In certain embodiments, the disease treated with theantibodies, antibody fragments (e.g., antigen binding fragments), andantibody drug conjugates of the invention is a cancer. Examples ofdiseases which can be treated and/or prevented include, but are notlimited to, adrenocortical carcinoma, bladder cancer, bone cancer,breast cancer, central nervous system atypical teratoid/rhabdoid tumors,colon cancer, colorectal cancer, embryonal tumors, endometrial cancer,gastric cancer, head and neck cancer, hepatocellular cancer, Kaposisarcoma, liver cancer, non-small cell lung cancer, rectal cancer,rhabdomyosarcoma, small cell lung cancer, small intestine cancer, softtissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomachcancer, uterine cancer, vaginal cancer, vulvar cancer. In certainembodiments, the cancer is characterized by FGFR2 or FGFR4 expressingcells to which the antibodies, antibody fragments (e.g., antigen bindingfragments), and antibody drug conjugates of the invention binds.

The present invention provides for methods of treating cancer comprisingadministering a therapeutically effective amount of the antibodies,antibody fragments (e.g., antigen binding fragments), or antibody drugconjugates of the invention. In certain embodiments, the cancer is asolid cancer. In certain embodiments, the subject is a human.

In certain embodiments, the method of inhibiting tumor growth comprisesadministering to a subject a therapeutically effective amount of theantibodies, antibody fragments (e.g., antigen binding fragments), orantibody drug conjugates of the invention. In certain embodiments, thesubject is a human. In certain embodiments, the subject has a tumor orhas had a tumor removed.

In certain embodiments, the tumor expresses the FGFR2 or FGFR4 to whichthe anti-FGFR2/4 antibody binds. In certain embodiments, the tumoroverexpresses the human FGFR2 or FGFR4.

For the treatment of the disease, the appropriate dosage of theantibodies, antibody fragments (e.g., antigen binding fragments), orantibody drug conjugates of the present invention depends on variousfactors, such as the type of disease to be treated, the severity andcourse of the disease, the responsiveness of the disease, previoustherapy, patient's clinical history, and so on. The antibody or agentcan be administered one time or over a series of treatments lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved (e.g., reduction in tumorsize). Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the patient and will vary depending onthe relative potency of an individual antibody, antibody fragment (e.g.,antigen binding fragment), or antibody drug conjugates. In certainembodiments, dosage is from 0.01 mg to 10 mg (e.g., 0.01 mg, 0.05 mg,0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 7 mg, 8 mg, 9 mg, or 10mg) per kg of body weight, and can be given once or more daily, weekly,monthly or yearly. In certain embodiments, the antibody, antibodyfragment (e.g., antigen binding fragment), or antibody drug conjugate ofthe present invention is given once every two weeks or once every threeweeks. The treating physician can estimate repetition rates for dosingbased on measured residence times and concentrations of the drug inbodily fluids or tissues.

Combination Therapy

In certain instances, an antibody, antibody fragment (e.g., antigenbinding fragment), or antibody drug conjugate of the present inventionis combined with other therapeutic agents, such as other anti-canceragents, anti-allergic agents, anti-nausea agents (or anti-emetics), painrelievers, cytoprotective agents, and combinations thereof.

General Chemotherapeutic agents considered for use in combinationtherapies include anastrozole (Arimidex®), bicalutamide (Casodex®),bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection(Busulfex®), capecitabine (Xeloda®),N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®),carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®),cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®),cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomeinjection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin(Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®),daunorubicin citrate liposome injection (DaunoXome®), dexamethasone,docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®),etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil(Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine(difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®),ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®),leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine(Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®),mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin,polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate(Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine(Tirazone®), topotecan hydrochloride for injection (Hycamptin®),vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®).

In one embodiment, an antibody, antibody fragment (e.g., antigen bindingfragment), or antibody drug conjugate of the present invention iscombined in a pharmaceutical combination formulation, or dosing regimenas combination therapy, with a second compound having anti-cancerproperties. The second compound of the pharmaceutical combinationformulation or dosing regimen can have complementary activities to theantibody or immunoconjugate of the combination such that they do notadversely affect each other. For example, an antibody, antibody fragment(e.g., antigen binding fragment), or antibody drug conjugate of thepresent invention can be administered in combination with, but notlimited to, a chemotherapeutic agent, a tyrosine kinase inhibitor, a FGFdownstream signaling pathway inhibitor, IAP inhibitors, Bcl2 inhibitors,Mcl1 inhibitors, and other FGFR2 inhibitors.

The term “pharmaceutical combination” as used herein refers to either afixed combination in one dosage unit form, or non-fixed combination or akit of parts for the combined administration where two or moretherapeutic agents may be administered independently at the same time orseparately within time intervals, especially where these time intervalsallow that the combination partners show a cooperative, e.g. synergisticeffect.

The term “combination therapy” refers to the administration of two ormore therapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients. Alternatively, such administration encompassesco-administration in multiple, or in separate containers (e.g.,capsules, powders, and liquids) for each active ingredient. Powdersand/or liquids may be reconstituted or diluted to a desired dose priorto administration. In addition, such administration also encompasses useof each type of therapeutic agent in a sequential manner, either atapproximately the same time or at different times. In either case, thetreatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

The combination therapy can provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect can be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect can be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e., serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

In one aspect, the present invention provides a method of treatingcancer by administering to a subject in need thereof an antibody drugconjugate of the present invention in combination with one or moretyrosine kinase inhibitors, including but not limited to, EGFRinhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors, and Metinhibitors.

For example, tyrosine kinase inhibitors include but are not limited to,Erlotinib hydrochloride (Tarceva®); Linifanib(N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N′-(2-fluoro-5-methylphenyl)urea,also known as ABT 869, available from Genentech); Sunitinib malate(Sutent®); Bosutinib(4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile,also known as SKI-606, and described in U.S. Pat. No. 6,780,996);Dasatinib (Sprycel®); Pazopanib (Votrient®); Sorafenib (Nexavar®);Zactima (ZD6474); and Imatinib or Imatinib mesylate (Gilvec® andGleevec®).

Epidermal growth factor receptor (EGFR) inhibitors include but are notlimited to, Erlotinib hydrochloride (Tarceva®), Gefitnib (Iressa®);N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide,Tovok®); Vandetanib (Caprelsa®); Lapatinib (Tykerb®);(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol(BMS690514); Canertinib dihydrochloride (CI-1033);6-[4-[(4-Ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-Pyrrolo[2,3-d]pyrimidin-4-amine(AEE788, CAS 497839-62-0); Mubritinib (TAK165); Pelitinib (EKB569);Afatinib (BIBW2992); Neratinib (HKI-272);N-[4-[[1-[(3-Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamicacid, (3 S)-3-morpholinylmethyl ester (BMS599626);N-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine(XL647, CAS 781613-23-8); and4-[4-[[(1R)-1-Phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol(PKI166, CAS 187724-61-4).

EGFR antibodies include but are not limited to, Cetuximab (Erbitux®);Panitumumab (Vectibix®); Matuzumab (EMD-72000); Trastuzumab(Herceptin®); Nimotuzumab (hR3); Zalutumumab; TheraCIM h-R3; MDX0447(CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1).

Human Epidermal Growth Factor Receptor 2 (HER2 receptor) (also known asNeu, ErbB-2, CD340, or p185) inhibitors include but are not limited to,Trastuzumab (Herceptin®); Pertuzumab (Omnitarg®); Neratinib (HKI-272,(2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide,and described PCT Publication No. WO 05/028443); Lapatinib or Lapatinibditosylate (Tykerb®);(3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol(BMS690514);(2E)-N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide(BIBW-2992, CAS 850140-72-6);N-[4-[[1-[(3-Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester(BMS 599626, CAS 714971-09-2); Canertinib dihydrochloride (PD183805 orCI-1033); andN-(3,4-Dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine(XL647, CAS 781613-23-8).

HER3 inhibitors include but are not limited to, LJM716, MM-121, AMG-888,RG7116, REGN-1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.

MET inhibitors include but are not limited to, Cabozantinib (XL184, CAS849217-68-1); Foretinib (GSK1363089, formerly XL880, CAS 849217-64-7);Tivantinib (ARQ197, CAS 1000873-98-2);1-(2-Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide(AMG 458); Cryzotinib (Xalkori®, PF-02341066);(3Z)-5-(2,3-Dihydro-1H-indol-1-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-1,3-dihydro-2H-indol-2-one(SU11271);(3Z)—N-(3-Chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N-methyl-2-oxoindoline-5-sulfonamide(SU11274);(3Z)—N-(3-Chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholin-4-ylpropyl)-1H-pyrrol-2-yl]methylene}-N-methyl-2-oxoindoline-5-sulfonamide(SU11606);6-[Difluoro[6-(1-methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]-quinoline(JNJ38877605, CAS 943540-75-8);2-[4-[1-(Quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl]-1H-pyrazol-1-yl]ethanol(PF04217903, CAS 956905-27-4);N-((2R)-1,4-Dioxan-2-ylmethyl)-N-methyl-N′-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide(MK2461, CAS 917879-39-1);6-[[6-(1-Methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]thio]-quinoline(SGX523, CAS 1022150-57-7); and(3Z)-5-[[(2,6-Dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2R)-2-(-pyrrolidinylmethyl)-1-pyrrolidinyl]carbonyl]-1H-pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one(PHA665752, CAS 477575-56-7).

IGF1R inhibitors include but are not limited to, BMS-754807, XL-228,OSI-906, GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and B1836845. See e.g., Yee, JNCI, 104; 975 (2012) forreview.

In another aspect, the present invention provides a method of treatingcancer by administering to a subject in need thereof an antibody drugconjugate of the present invention in combination with one or more FGFdownstream signaling pathway inhibitors, including but not limited to,MEK inhibitors, Braf inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors,and also mTor.

For example, mitogen-activated protein kinase (MEK) inhibitors includebut are not limited to, XL-518 (also known as GDC-0973, Cas No.1029872-29-4, available from ACC Corp.);2-[(2-Chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide(also known as CI-1040 or PD184352 and described in PCT Publication No.WO2000035436);N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide(also known as PD0325901 and described in PCT Publication No.WO2002006213);2,3-Bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also knownas U0126 and described in U.S. Pat. No. 2,779,780);N-[3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)-2,3-dihydroxypropyl]-cyclopropanesulfonamide(also known as RDEA119 or BAY869766 and described in PCT Publication No.WO2007014011);(3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione](also known as E6201 and described in PCT Publication No. WO2003076424);2′-Amino-3′-methoxyflavone (also known as PD98059 available from BiaffinGmbH & Co., KG, Germany); Vemurafenib (PLX-4032, CAS 918504-65-1);(R)-3-(2,3-Dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione(TAK-733, CAS 1035555-63-5); Pimasertib (AS-703026, CAS 1204531-26-9);and Trametinib dimethyl sulfoxide (GSK-1120212, CAS 1204531-25-80).

Phosphoinositide 3-kinase (PI3K) inhibitors include but are not limitedto,4-[2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine(also known as GDC 0941 and described in PCT Publication Nos. WO09/036082 and WO 09/055730);2-Methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile(also known as BEZ 235 or NVP-BEZ 235, and described in PCT PublicationNo. WO 06/122806);4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine(also known as BKM120 or NVP-BKM120, and described in PCT PublicationNo. WO2007/084786); Tozasertib (VX680 or MK-0457, CAS 639089-54-6);(5Z)-5-[[4-(4-Pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione(GSK1059615, CAS 958852-01-2);(1E,4S,4aR,5R,6aS,9aR)-5-(Acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione(PX866, CAS 502632-66-8); and 8-Phenyl-2-(morpholin-4-yl)-chromen-4-one(LY294002, CAS 154447-36-6).

mTor include but are not limited to, Temsirolimus (Torisel®);Ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4,9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383); Everolimus (Afinitor® or RAD001);Rapamycin (AY22989, Sirolimus®); Simapimod (CAS 164301-51-3);(5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol(AZD8055);2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one(PF04691502, CAS 1013101-36-4); andN²-[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-,inner salt (SF1126, CAS 936487-67-1).

In yet another aspect, the present invention provides a method oftreating cancer by administering to a subject in need thereof anantibody drug conjugate of the present invention in combination with oneor more pro-apoptotics, including but not limited to, IAP inhibitors,Bcl2 inhibitors, MC11 inhibitors, Trail agents, Chk inhibitors.

For examples, IAP inhibitors include but are not limited to, LCL161,GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAPinhibitors include but are not limited to those disclosed inWO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118,WO 06/017295, and WO08/134679, all of which are incorporated herein byreference.

BCL-2 inhibitors include but are not limited to,4-[4-[[2-(4-Chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide(also known as ABT-263 and described in PCT Publication No. WO09/155386); Tetrocarcin A; Antimycin; Gossypol ((-)BL-193); Obatoclax;Ethyl-2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2-oxoethyl)-4Hchromone-3-carboxylate(HA14-1); Oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-Gossypolacetic acid (AT-101);4-[4-[(4′-Chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide(ABT-737, CAS 852808-04-9); and Navitoclax (ABT-263, CAS 923564-51-6).

Proapoptotic receptor agonists (PARAs) including DR4 (TRAILR1) and DR5(TRAILR2), including but are not limited to, Dulanermin (AMG-951,RhApo2L/TRAIL); Mapatumumab (HRS-ETR1, CAS 658052-09-6); Lexatumumab(HGS-ETR2, CAS 845816-02-6); Apomab (Apomab®); Conatumumab (AMG655, CAS896731-82-1); and Tigatuzumab (CS1008, CAS 946415-34-5, available fromDaiichi Sankyo).

Checkpoint Kinase (CHK) inhibitors include but are not limited to,7-Hydroxystaurosporine (UCN-01);6-Bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(3R)-3-piperidinyl-pyrazolo[1,5-a]pyrimidin-7-amine(SCH900776, CAS 891494-63-6);5-(3-Fluorophenyl)-3-ureidothiophene-2-carboxylic acidN—[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-1-Azabicyclo[2.2.2]oct-3-yl)amino]-3-(1H-benzimidazol-2-yl)-6-chlnolin-2(1H)-one (CHIR 124, CAS 405168-58-3); 7-Aminodactinomycin(7-AAD), Isogranulatimide, debromohymenialdisine;N-[5-Bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]-phenyl]-N′-(5-methyl-2-pyrazinyl)urea(LY2603618, CAS 911222-45-2); Sulforaphane (CAS 4478-93-7,4-Methylsulfinylbutyl isothiocyanate);9,10,11,12-Tetrahydro-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-1,3(2H)-dione(SB-218078, CAS 135897-06-2); and TAT-S216A (YGRKKRRQRRRLYRSPAMPENL),(SEQ ID NO: 143), and CBP501 ((d-Bpa)sws(d-Phe-F5)(d-Cha)rrrqrr).

In one aspect, the present invention provides a method of treatingcancer by administering to a subject in need thereof an antibody drugconjugate of the present invention in combination with one or more FGFRinhibitors. For example, FGFR inhibitors include but are not limited to,Brivanib alaninate (BMS-582664,(S)—((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-J][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate);Vargatef (BIBF1120, CAS 928326-83-4); Dovitinib dilactic acid (TKI258,CAS 852433-84-2);3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea(BGJ398, CAS 872511-34-7); Danusertib (PHA-739358); andN-[2-[[4-(Diethylamino)butyl]amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N′-(1,1-dimethylethyl)-urea (PD173074, CAS219580-11-7). In a specific embodiment, the present invention provides amethod of treating cancer by administering to a subject in need thereofan antibody drug conjugate of the present invention in combinationanother FGFR2 inhibitor, such as3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methylurea(also known as BGJ-398); or 4-amino-5-fluoro-3-(5-(4-methylpiperazin1-yl)-1H-benzo[d]imidazole-2-yl)quinolin-2(1H)-one (also known asdovitinib or TKI-258). AZD4547 (Gavine et al., 2012, Cancer Research 72,2045-56, N-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3R,5S)-diemthylpiperazin-1-yl)benzamide), Ponatinib (AP24534; Gozgit et al.,2012, Mol Cancer Ther., 11; 690-99;3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide,CAS 943319-70-8)

Pharmaceutical Compositions

To prepare pharmaceutical or sterile compositions includingimmunoconjugates, the immunoconjugates of the invention are mixed with apharmaceutically acceptable carrier or excipient. The compositions canadditionally contain one or more other therapeutic agents that aresuitable for treating or preventing cancer (breast cancer, colorectalcancer, lung cancer, multiple myeloma, ovarian cancer, liver cancer,gastric cancer, pancreatic cancer, acute myeloid leukemia, chronicmyeloid leukemia, osteosarcoma, squamous cell carcinoma, peripheralnerve sheath tumors schwannoma, head and neck cancer, bladder cancer,esophageal cancer, Barretts esophageal cancer, glioblastoma, clear cellsarcoma of soft tissue, malignant mesothelioma, neurofibromatosis, renalcancer, melanoma, prostate cancer, benign prostatic hyperplasia (BPH),gynacomastica, and endometriosis).

Formulations of therapeutic and diagnostic agents can be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions, lotions, or suspensions (see, e.g., Hardman et al., Goodmanand Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y., 2001; Gennaro, Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y., 2000; Avis,et al. (eds.), Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, N.Y., 1993; Lieberman, et al. (eds.), PharmaceuticalDosage Forms: Tablets, Marcel Dekker, N.Y., 1990; Lieberman, et al.(eds.) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker,N.Y., 1990; Weiner and Kotkoskie, Excipient Toxicity and Safety, MarcelDekker, Inc., New York, N.Y., 2000).

In a specific embodiment, the clinical service form (CSF) of theantibody drug conjugates of the present invention is a lyophilisate invial containing the ADC, sodium succinate, and polysorbate 20. Thelyophilisate can be reconstitute with water for injection, the solutioncomprises the ADC, sodium succinate, sucrose, and polysorbate 20 at a pHof about 5.0. For subsequent intravenous administration, the obtainedsolution will usually be further diluted into a carrier solution.

Selecting an administration regimen for a therapeutic depends on severalfactors, including the serum or tissue turnover rate of the entity, thelevel of symptoms, the immunogenicity of the entity, and theaccessibility of the target cells in the biological matrix. In certainembodiments, an administration regimen maximizes the amount oftherapeutic delivered to the patient consistent with an acceptable levelof side effects. Accordingly, the amount of biologic delivered dependsin part on the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak,Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK, 1996;Kresina (ed.), Monoclonal Antibodies, Cytokines and Arthritis, MarcelDekker, New York, N.Y., 1991; Bach (ed.), Monoclonal Antibodies andPeptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.,1993; Baert et al., New Engl. J. Med. 348:601-608, 2003; Milgrom et al.,New Engl. J. Med. 341:1966-1973, 1999; Slamon et al., New Engl. J. Med.344:783-792, 2001; Beniaminovitz et al., New Engl. J. Med. 342:613-619,2000; Ghosh et al., New Engl. J. Med. 348:24-32, 2003; Lipsky et al.,New Engl. J. Med. 343:1594-1602, 2000).

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors known in the medical arts.

Compositions comprising antibodies or fragments thereof of the inventioncan be provided by continuous infusion, or by doses at intervals of,e.g., one day, one week, or 1-7 times per week, once every other week,once every three weeks, once every four weeks, once every five weeks,once every six weeks, once every seven weeks, or once very eight weeks.Doses may be provided intravenously, subcutaneously, topically, orally,nasally, rectally, intramuscular, intracerebrally, or by inhalation. Aspecific dose protocol is one involving the maximal dose or dosefrequency that avoids significant undesirable side effects.

For the immunoconjugates of the invention, the dosage administered to apatient may be 0.0001 mg/kg to 100 mg/kg of the patient's body weight.The dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg,0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's bodyweight. The dosage of the antibodies or fragments thereof of theinvention may be calculated using the patient's weight in kilograms (kg)multiplied by the dose to be administered in mg/kg.

Doses of the immunoconjugates the invention may be repeated and theadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months. In a specific embodiment, doses of theimmunoconjugates of the invention are repeated every 3 weeks.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method, route and dose of administration and the severityof side effects (see, e.g., Maynard et al., A Handbook of SOPs for GoodClinical Practice, Interpharm Press, Boca Raton, Fla., 1996; Dent, GoodLaboratory and Good Clinical Practice, Urch Publ., London, UK, 2001).

The route of administration may be by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or by sustained release systems or animplant (see, e.g., Sidman et al., Biopolymers 22:547-556, 1983; Langeret al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem. Tech.12:98-105, 1982; Epstein et al., Proc. Natl. Acad. Sci. USA82:3688-3692, 1985; Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034, 1980; U.S. Pat. Nos. 6,350,466 and 6,316,024). Wherenecessary, the composition may also include a solubilizing agent or alocal anesthetic such as lidocaine to ease pain at the site of theinjection, or both. In addition, pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320,5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entirety.

A composition of the present invention may also be administered via oneor more routes of administration using one or more of a variety ofmethods known in the art. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. Selected routes of administration for theimmunoconjugates of the invention include intravenous, intramuscular,intradermal, intraperitoneal, subcutaneous, spinal or other parenteralroutes of administration, for example by injection or infusion.Parenteral administration may represent modes of administration otherthan enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Alternatively, a composition of theinvention can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically. Inone embodiment, the immunoconjugates of the invention is administered byinfusion. In another embodiment, the immunoconjugates of the inventionis administered subcutaneously.

If the immunoconjugates of the invention are administered in acontrolled release or sustained release system, a pump may be used toachieve controlled or sustained release (see Langer, supra; Sefton, CRCCrit. Ref Biomed. Eng. 14:20, 1987; Buchwald et al., Surgery 88:507,1980; Saudek et al., N. Engl. J. Med. 321:574, 1989). Polymericmaterials can be used to achieve controlled or sustained release of thetherapies of the invention (see e.g., Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., 1974;Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, New York, 1984; Ranger and Peppas, J.Macromol. Sci. Rev. Macromol. Chem. 23:61, 1983; see also Levy et al.,Science 228:190, 1985; During et al., Ann. Neurol. 25:351, 1989; Howardet al., J. Neurosurg. 7 1:105, 1989; U.S. Pat. No. 5,679,377; U.S. Pat.No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S.Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT PublicationNo. WO 99/20253. Examples of polymers used in sustained releaseformulations include, but are not limited to, poly(2-hydroxy ethylmethacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In oneembodiment, the polymer used in a sustained release formulation isinert, free of leachable impurities, stable on storage, sterile, andbiodegradable. A controlled or sustained release system can be placed inproximity of the prophylactic or therapeutic target, thus requiring onlya fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138, 1984).

Controlled release systems are discussed in the review by Langer,Science 249:1527-1533, 1990). Any technique known to one of skill in theart can be used to produce sustained release formulations comprising oneor more immunoconjugates of the invention. See, e.g., U.S. Pat. No.4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698,Ning et al., Radiotherapy & Oncology 39:179-189, 1996; Song et al., PDAJournal of Pharmaceutical Science & Technology 50:372-397, 1995; Cleeket al., Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, 1997;and Lam et al., Proc. Int'l. Symp. Control Rel. Bioact. Mater.24:759-760, 1997, each of which is incorporated herein by reference intheir entirety.

If the immunoconjugates of the invention are administered topically,they can be formulated in the form of an ointment, cream, transdermalpatch, lotion, gel, spray, aerosol, solution, emulsion, or other formwell-known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms,19th ed., Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topicaldosage forms, viscous to semi-solid or solid forms comprising a carrieror one or more excipients compatible with topical application and havinga dynamic viscosity, in some instances, greater than water are typicallyemployed. Suitable formulations include, without limitation, solutions,suspensions, emulsions, creams, ointments, powders, liniments, salves,and the like, which are, if desired, sterilized or mixed with auxiliaryagents (e.g., preservatives, stabilizers, wetting agents, buffers, orsalts) for influencing various properties, such as, for example, osmoticpressure. Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, in some instances, incombination with a solid or liquid inert carrier, is packaged in amixture with a pressurized volatile (e.g., a gaseous propellant, such asfreon) or in a squeeze bottle. Moisturizers or humectants can also beadded to pharmaceutical compositions and dosage forms if desired.Examples of such additional ingredients are well-known in the art.

If the compositions comprising the immunoconjugates are administeredintranasally, it can be formulated in an aerosol form, spray, mist or inthe form of drops. In particular, prophylactic or therapeutic agents foruse according to the present invention can be conveniently delivered inthe form of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant (e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are known in the art (see, e.g., Hardman et al., (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10.sup.th ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.). An effective amount of therapeutic may decreasethe symptoms by at least 10%; by at least 20%; at least about 30%; atleast 40%, or at least 50%.

Additional therapies (e.g., prophylactic or therapeutic agents), whichcan be administered in combination with the immunoconjugates of theinvention may be administered less than 5 minutes apart, less than 30minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hoursto about 4 hours apart, at about 4 hours to about 5 hours apart, atabout 5 hours to about 6 hours apart, at about 6 hours to about 7 hoursapart, at about 7 hours to about 8 hours apart, at about 8 hours toabout 9 hours apart, at about 9 hours to about 10 hours apart, at about10 hours to about 11 hours apart, at about 11 hours to about 12 hoursapart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart,24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120hours apart from the immunoconjugates of the invention. The two or moretherapies may be administered within one same patient visit.

In certain embodiments, the immunoconjugates of the invention can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., Ranade, (1989)J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folateor biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);antibodies (Bloeman et al., (1995) FEBS Lett. 357:140; Owais et al.,(1995) Antimicrob. Agents Chemother. 39:180); surfactant protein Areceptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134); p 120(Schreier et al, (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273.

The invention provides protocols for the administration ofpharmaceutical composition comprising immunoconjugates of the inventionalone or in combination with other therapies to a subject in needthereof. The therapies (e.g., prophylactic or therapeutic agents) of thecombination therapies of the present invention can be administeredconcomitantly or sequentially to a subject. The therapy (e.g.,prophylactic or therapeutic agents) of the combination therapies of thepresent invention can also be cyclically administered. Cycling therapyinvolves the administration of a first therapy (e.g., a firstprophylactic or therapeutic agent) for a period of time, followed by theadministration of a second therapy (e.g., a second prophylactic ortherapeutic agent) for a period of time and repeating this sequentialadministration, i.e., the cycle, in order to reduce the development ofresistance to one of the therapies (e.g., agents) to avoid or reduce theside effects of one of the therapies (e.g., agents), and/or to improve,the efficacy of the therapies.

The therapies (e.g., prophylactic or therapeutic agents) of thecombination therapies of the invention can be administered to a subjectconcurrently.

The term “concurrently” is not limited to the administration oftherapies (e.g., prophylactic or therapeutic agents) at exactly the sametime, but rather it is meant that a pharmaceutical compositioncomprising antibodies or fragments thereof the invention areadministered to a subject in a sequence and within a time interval suchthat the antibodies of the invention can act together with the othertherapy(ies) to provide an increased benefit than if they wereadministered otherwise. For example, each therapy may be administered toa subject at the same time or sequentially in any order at differentpoints in time; however, if not administered at the same time, theyshould be administered sufficiently close in time so as to provide thedesired therapeutic or prophylactic effect. Each therapy can beadministered to a subject separately, in any appropriate form and by anysuitable route. In various embodiments, the therapies (e.g.,prophylactic or therapeutic agents) are administered to a subject lessthan 15 minutes, less than 30 minutes, less than 1 hour apart, at about1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hoursto about 3 hours apart, at about 3 hours to about 4 hours apart, atabout 4 hours to about 5 hours apart, at about 5 hours to about 6 hoursapart, at about 6 hours to about 7 hours apart, at about 7 hours toabout 8 hours apart, at about 8 hours to about 9 hours apart, at about 9hours to about 10 hours apart, at about 10 hours to about 11 hoursapart, at about 11 hours to about 12 hours apart, 24 hours apart, 48hours apart, 72 hours apart, or 1 week apart. In other embodiments, twoor more therapies (e.g., prophylactic or therapeutic agents) areadministered to a within the same patient visit.

The prophylactic or therapeutic agents of the combination therapies canbe administered to a subject in the same pharmaceutical composition.Alternatively, the prophylactic or therapeutic agents of the combinationtherapies can be administered concurrently to a subject in separatepharmaceutical compositions. The prophylactic or therapeutic agents maybe administered to a subject by the same or different routes ofadministration.

EXAMPLES

The following examples and intermediates serve to illustrate theinvention without limiting the scope thereof.

Example 1: Screening for Anti-FGFR2/4 Antibodies Cell Lines

Ba/F3 cells were purchased from DSMZ, KatoIII, SNU16, SNU5 and HCl-H716cells were purchased from the American Type Culture Collection (ATCC)and NUGC3 cells were obtained from the Japanese Collection of ResearchBioresources (JCRB) Cell Bank. All cell lines were routinely cultured inappropriate growth medium supplemented with fetal bovine serum (FBS) asrecommended by their respective suppliers.

Generation of Recombinant Human, Cyno, Mouse and Rat FGFR Vectors

Human, mouse and rat FGFR extracellular domains were gene synthesizedbased on amino acid sequences from the GenBank or Uniprot databases (seeTable 2). Cynomolgus FGFR2 and 4 ECD cDNA templates were genesynthesized based on amino acid sequences information generated usingmRNA from various cyno tissues (e.g. Zyagen Laboratories; Table 3). Allsynthesized DNA fragments were cloned into appropriate expressionvectors e.g. CMV based vectors or pCDNA3.1. with C-terminal tags toallow for purification.

TABLE 2 Generation of FGFR expression vectors. Amino acid numbering isbased on the accession numbers or seq IDs provided in the table NameDescription Accession Number SEQ ID NO Human FGFR1 Human FGFR1 isoformIIIb, NM 015850 121 IIIb D1-3 residues 22-373-TAG Human FGFR2 HumanFGFR2 isoform IIIb, NM_022970/P21802-3 122 IIIb D1-3 residues 22-378-TAGHuman FGFR3 Human FGFR3 isoform IIIb, NM_000142 123 IIIb D1-3 residues23-377-TAG Human FGFR4 Human FGFR4 extracellular domain, NM_002011;P22455 144 ECD residues 22-369-TAG Human FGFR2 Human FGFR2 isoform IIIc,NM_022970/P21802-1 125 IIIc D1-3 residues 22-378-TAG CynomolgusCynomolgus monkey FGFR2 Not applicable. 126 monkey FGFR2 isoform IIIb,residues 22-378-TAG IIIb D1-3 Mouse FGFR2 Mouse FGFR2 isoform IIIb,residues NM_010207 127 IIIb D1-3 22-378-TAG Rat FGFR2 IIIb Rat FGFR2isoform IIIb, residues XM_001079450 128 D1-3 41-397-TAG Rat FGFR4 RatFGFR4, residues 17-367-TAG NM_001109904 129 Cynomolgus Cynomolgus monkeyFGFR4, Not applicable. 130 monkey FGFR4 residues 17-366-TAG Mouse FGFR4Mouse FGFR4, residues 17-366- Mouse NM_008011 131 TAG

TABLE 3 Sequences of cynomolgus FGFR2 and FGFR4 proteinsAmino acid sequence SEQ Construct in one letter code ID NO CynomolgusMETDTLLLWVLLLWVPGSTGRPSFSLV 126 monkey FGFR2 EDTTLEPEEPPTKYQISQPEVYVAAPGIIIb D1-3 ESLEVRCLLKDAAVISWTKDGVHLGPN NRTVLIGEYLQIKGATPRDSGLYACTATRTVDSETWYFMVNVTDAISSGDDEDD TDGAEDFVSENGNNKRAPYWTNTEKMEKRLHAVPAANTVKFRCPAGGNPTPTMR WLKNGKEFKQEHRIGGYKVRNQHWSLIMESVVPSDKGNYTCVVENEYGSINHTY HLDVVERSPHRPILQAGLPANASTVVGGDVEFVCKVYSDAQPHIQWIKHVEKNG SKYGPDGLPYLKVLKHSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQS AWLTVLPKQQAPGREKEITASPDYLEKLEFRHDSGLNDIFEAQKIEWHE Cynomolgus METDTLLLWVLLLWVPGSTGLEASEEV 130monkey FGFR4 ELEPCLAPSMEQQEQELTVALGQPVRL CCGRAERGGHWYKEGSRLAPAGRVRGWRGRLEIASFLPEDAGRYLCLARASMIV LQNVTLTIDDSLTSSNDDEDPQSHRDSSNGHIYPQQAPYWTHPQRMEKKLHAVP AGNTVKFRCPAAGNPTPTIRWLKDGQAFHGENRIGGIRLRHQHWSLVMESVVPS DRGTYTCLVENAVGSIRYNYLLDVLERSPHRPILQAGLPANTTAVVGSDVELLC KVYSDAQPHIQWLKHIVINGSSFGADGFPYVQVLKTADINSSEVEVLYLRNVSA EDAGEYTCLAGNSIGLSYQSAWLTVLPEEDLTWTAATPEARYTDKLEFRHDSGL NDIFEAQKIEWHE

Expression of Recombinant FGFR Proteins

The desired FGFR recombinant proteins were expressed in HEK293 derivedcell lines (293T) previously adapted to suspension culture and grown ina mix of serum-free medium, 50% HyClone SFM4Transfx-293 withoutL-glutamine (HyClone) and 50% FreeStyle-293 (Gibco). Both small scaleand large scale protein production were via transient transfection andwas performed in multiple shaker flasks (Nalgene), up to 1 L each, withPolyethylenimine (PEI, 25K, AlfaAesar) as a plasmid carrier. Whenbiotinylated proteins were to be expressed via the C-terminal Avi tag, aplasmid carrying the gene for BirA enzyme were included at the ratio of1:2 to the ECD protein plasmid. Total DNA and Polyethylenimine was usedat a ratio of 1:3 (w:w). DNA to culture ratio was 1 mg/L. The cellculture supernatants were harvested 6 days post transfection,centrifuged and sterile filtered prior to purification.

Tagged Protein Purification

Recombinant tagged FGFR2 and FGFR4 ECD proteins (e.g., APP-FGFR2 andAPP-FGFR4 ECD) were purified by collecting the cell culture supernatant.An anti-APP column was prepared by coupling an anti-APP monoclonalantibody to CNBr activated Sepharose 4B at a final ratio of 10 mgantibody per mL of resin. Expression supernatant was applied to ananti-APP column at a flow rate of 1-2 mL/minute or by gravity flow.After base-line washing with PBS, bound material was eluted with 100 mMglycine (pH 2.7) and immediately dialyzed against PBS overnight and thensterile filtered. Protein concentrations were determined by measuringthe absorbance at 280 nm and converting using the protein extinctioncoefficient calculated based on individual protein's amino acidsequences. The purified protein was then characterized by SDS-PAGE,analytical size exclusion chromatography (HPLC-SEC). For those that hadmore than >10% aggregates in the affinity purified preparation, a secondstep SEC purification were performed followed by confirmationcharacterizations.

Generation of Ba/F3 FGFR2 Cell Lines

To generate these cells, human FRGR2-IIIb-C3 (NM_022970) was cloned intoa pENTR TOPOD vector (Invitrogen catalog #K2400-20) and then into apLenti6 DEST vector (Invitrogen catalog #V49610) under a CMV promoter.Virus was then packaged and Ba/F3 cells (DSMZ catalog #ACC300) were spininfected with the lentivirus, followed by treatment with 20 μgblasticidin (e.g. Invitrogen, catalog # A11139-03 or Cellgro, catalog#30-100-RB) and selection with IL3 (R&D systems catalog #403-ml-010) for5 days in a tissue culture incubator at 37° C. and 5% CO₂. Survivingcells were then grown in medium (RPMI (Invitrogen catalog #11875-093)supplemented with 10% FBS (Clontech catalog #631101, 2 μg/ml heparin(Sigma, catalog # H3149) and 20 μg/ml blasticidin (e.g. Invitrogen,catalog # A11139-03 or Cellgro, catalog #30-100-RB)) but without IL3 for1 month. Surviving cells were then dilution cloned to generate clonalcell populations which were then used for subsequent studies.

HuCAL PLATINUM® Pannings

For the selection of antibidies recognizing human FGFR2, multiplepanning strategies were utilized. Therapeutic antibodies against humanFGFR proteins were generated by the selection of clones that bound toFGFR2 using as a source of antibody variant proteins a commerciallyavailable phage display library, the Morphosys HuCAL PLATINUM® library.The phagemid library is based on the HuCAL® concept (Knappik et al.,2000, J Mol Biol 296: 57-86) and employs the CysDisplay™ technology fordisplaying the Fab on the phage surface (WO01/05950).

For the isolation of anti-FGFR2 antibodies, several different panningstrategies were employed, using solid phase, solution, whole cell anddifferential whole cell approaches.

Solid Phase Panning on Recombinant FGFR2

Prior to the antigen selection process a coating check ELISA wasperformed to determine the optimal coating concentration for theantigen. Different recombinant FGFR2 proteins with various tags wereused in the solid phase panning approach by coating on Maxisorp™ plates(Nunc) either via passive adsoption or via capture antibodies targetingthe respective tag of the antigen. Alternatively Reacti-Bind™NeutrAvidin™-coated Polystyrene Strip Plates (Pierce) were used tocapture biotinylated FGFR2 antigen. An appropriate number (dependent onthe number of sub-library pools) of wells of a 96-well Maxisorp™ plate(Nunc) were coated with antigen overnight at 4° C. The coated wells wereblocked with PBS (phosphate buffered saline)/5% milk powder. For eachpanning, about HuCAL PLATINUM® phage-antibodies were blocked. After theblocking procedure, pre-blocked phage mix was added to each antigencoated and blocked well and incubated for 2 hours (h) at roomtemperature (RT) on a microtiter plate (MTP) shaker. Afterwards,unspecific bound phage was washed off by several washing steps. Forelution of specifically bound phage, 25 mM DTT (Dithiothreitol) wasadded for 10 minutes (min) at RT. The DTT eluates were used forinfection of E. coli (Escherichia coli) TG-1 cells. After infection, thebacteria were plated on LB (Jysogeny broth)/Cam (chloramphenicol) agarplates and incubated overnight at 30° C. Colonies were scraped off theplates and were used for phage rescue, polyclonal amplification ofselected clones, and phage production. Each FGFR2 solid phase panningstrategy comprised individual rounds of panning and contained uniqueantigens, antigen concentrations, buffer compositions and washingstringencies.

Solution Panning on Recombinant FGFR2 with Streptavidin-Coupled MagneticBeads

A prerequisite for solution panning approaches was the biotinylation ofthe antigen and confirmation of retained activity of biotinylatedantigen. During solution panning, the Fab displaying phage and thebiotinylated antigen were incubated in solution which facilitated theaccessibility of the antigen by the phage

For each phage pool, Streptavidin beads (Dynabeads® M-280 Streptavidin;Invitrogen) were blocked in 1× Chemiblocker. In parallel, for eachpanning, about HuCAL PLATINUM® phage-antibodies were blocked with anequal volume of 2× Chemiblocker/0.1% Tween20. Then, a certainconcentration of biotinylated antigen (e.g. 100 nM) was added to thepre-adsorbed and blocked phage particles and incubated for 1-2 h at RTon a rotator. The phage-antigen complexes were captured using blockedStreptavidin beads and phage particles bound to the Streptavidin beadswere collected with a magnetic separator. Unspecific bound phages werewashed off by several washing steps. For elution of specifically boundphage from Streptavidin beads, 25 mM DTT were added for 10 min at RT.The DTT eluate was processed as described for the solid phase pannings.

Each FGFR2 solution phase panning strategy comprised individual roundsof panning and contained unique antigens, antigen concentrations andwashing stringencies.

Whole Cell Panning on FGFR2 Overexpressing Cells

For each cell panning, HuCAL PLATINUM® phage-antibodies were pre-blockedin PBS/FCS In parallel, 0.5-1.0×10⁷ target cells per phage pool (Ba/F3cells stably transfected with FGFR2, Kato-III, SNU16, H716) showingoverexpression of FGFR2 were resuspended in PBS/FCS on ice.

The blocked target cells were spun down, re-suspended in the pre-blockedphage particles and incubated for 2 h at 4° C. on a rotator. Thephage-cell complexes were washed in PBS/FCS. Elution of specificallybound phage from target cells was performed by acidic elution withglycine buffer, pH 2.2. After centrifugation, the supernatant (eluate)was neutralized by adding unbuffered Tris. The final phage containingsupematant was used for infection of E. coli TG1 culture. The followingsteps were done as described under the section solid phase pannings.

In more detail, either whole cell pannings were performed, where eachpanning round was performed with cells or, alternatively, differentialwhole cell pannings were performed, meaning either cells or recombinantprotein was used in consecutive panning rounds. The selection rounds onrecombinant antigen were performed as described for solid phase orsolution pannings.

Maturation Pannings

In order to obtain FGFR2 specific antibodies with increased affinities,maturation pannings were performed (Prassler et al., 2009,Immunotherapy, 1: 571-583). For this purpose, standard pannings (solidphase and solution phase panning) were performed with different FGFR2antigens as described above.

After the panning, Fab-encoding fragments of phage derived pMORPH30®vector DNA were digested with distinct specific restriction enzymes togenerate either LCDR3 or HCDR2 matured libraries. The insertes werereplaced using TRIM™ technology (Vimekas et al., 1994, Nucleic AcidsResearch 22: 5600-5607).

The generated libraries were amplified and subjected to two more roundsof panning with increased washing stringency and reduced antigenconcentrations.

Subcloning and Microexpression of Selected Fab Fragments

To facilitate rapid expression of soluble Fab, the Fab encoding insertsof the selected HuCAL PLATINUM® phage were subcloned from pMORPH®30display vector into pMORPH®x11 expression vector pMORPH®x11_FH.

After transformation of E. coli TG1-F⁻ single clone expression andpreparation of periplasmic extracts containing HuCAL®-Fab fragments wereperformed as described previously (Rauchenberger et al., 2003 J BiolChem 278: 38194-38205).

ELISA Screening

Using ELISA screening, single Fab clones are identified from panningoutput for binding to the target antigen. Fabs are tested using Fabcontaining crude E. coli lysates.

Maxisorp™ (Nunc) 384 well plates were coated with FGFR antigens ofinterest (either via passive adsoption or via capure antibodiestargeting the respective tag of the antigen) in PBS at their previousdetermined saturation concentration. Alternatively, Reacti-Bind™NeutrAvidin-coated Polystyrene Strip Plates (Pierce) were used tocapture biotinylated FGFR2 antigen.

After blocking of plates with 5% skim milk powder in PBS, Fab-containingE. coli lysates were added. Binding of Fabs was detected by F(ab)₂specific goat anti-human IgG conjugated to alkaline phosphatase (diluted1:5000) using Attophos fluorescence substrate (Roche, catalog#11681982001). Fluorescence emission at 535 nm was recorded withexcitation at 430 nm.

FACS Screening (Fluorescence Activated Cell Sorting)

In FACS screening, single Fab clones binding to cell surface expressedantigen are identified from the panning output. Fabs are tested for cellbinding using Fab containing crude E. coli lysates.

In these studies, 100 μl of cell-suspension was transferred into a fresh96-well plate (resulting in 1×10⁵ cells/well). Target cell suspensioncontaining plates were centrifuged and the supernatant was discarded.The remaining cell pellets were re-suspended and 50 μl of Fab containingbacterial extracts was added to the corresponding wells.

Alternatively, the cell pellet was re-suspended and 50 μl FACS buffer(PBS, 3% FCS) and same volume of Fab containing bacterial extracts wasadded to the corresponding wells.

The cell-antibody suspensions were then incubated on ice for 1 hour.Following incubation, cells were spun down and washed three times with200 μl FACS buffer. After each washing step, cells were centrifuged andcarefully re-suspended.

Secondary detection antibody (PE conjugated goat anti human IgG;Dianova) was added and samples were incubate on ice and subsequentlywashed according to Fab incubation Fluorescence intensity was determinedin a FACSArray™ instrument.

Expression and Purification of HuCAL® Fab Fragments

Expression of Fab fragments was performed in E. coli TG1 F-cells.Cultures were shaken at 30° C. for 18 h. Cells were harvested anddisrupted using a combination of lysozyme and Bug Buster ProteinExtraction Reagent (Novagen, Germany). His6-tagged (SEQ ID NO: 142) Fabfragments were isolated via IMAC (Qiagen, Germany) and proteinconcentrations were determined by UV-spectrophotometry. The purity ofrepresentatively selected samples was analyzed in denaturing, reducing15% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis).

The homogeneity of Fab preparations was determined in native state bysize exclusion chromatography (HP-SEC) with calibration standards.

Conversion to IgG and IgG Expression

In order to express full length IgG, variable domain fragments of heavy(VH) and light chains (VL) were subcloned from Fab expression vectorsinto appropriate pMorph®_hIg vectors for human IgG1. Alternatively,eukaryotic HKB11 cells were transfected with pMORPH®4 expression vectorDNA. The cell culture supernatant was harvested 3 or 7 days posttransfection. After sterile filtration, the solution was subjected toProtein A affinity chromatography (MabSelect SURE, GE Healthcare) usinga liquid handling station. If not otherwise stated, buffer exchange wasperformed to 1× Dulbecco's PBS (pH 7.2, Invitrogen) and samples weresterile filtered (0.2 μm pore size). Protein concentrations weredetermined by UV-spectrophotometry and purity of IgGs was analyzed underdenaturing, reducing conditions using a Caliper Labchip System or inSDS-PAGE.

Bioassays

Anti-FGFR antibodies obtained following the panning processes describedabove were evaluated in the assays exemplified below:

BaF3/CMV-FGFRIIIB-C3 Cell Proliferation Assay

To determine the capacity of anti-FGFR2 antibodies to inhibitFGF1-dependent cell proliferation, a proliferation assay using anengineered Ba/F3 cell line (BaF3/CMV-FGFRIIIB-C3), which was stablytransduced with a chimera of full-length FGFR2IIIb receptor ECD andFGFR1-intracellular domain harboring the respective kinase domains.Addition of human FGF1 promoted cell proliferation in this cell line.

Cells were re-suspended in full-growth medium (RPMI+10% FCS+30 ng/mlFGF1+2 μg/ml Heparin+20 μg/ml Blasticidin) and seeded into flat-bottomedwhite 96-well assay plates (Corning Costar, #3903) at a cell density of8×10³ cells/well in 80 μl and incubated at 37° C. and 5% CO₂ overnight.

The next day, the HuCAL® antibodies (Fab or IgG) were diluted infull-growth medium to the desired concentrations (5 fold concentrated).20 μl of the antibody solutions were added to the seeded cells of theprevious day and the cells were cultivated for 72 hr. After 72 hr, 100μl Cell Titer-Glo Luminescent Cell Viability Assay Reagent (#G7571;Promega) was added to each well and incubated for 15 min at RT slightlyshaking with subsequent read-out of the luminescence in a illuminometer(GeniosPro, Tecan). For determination of the half maximal inhibitoryconcentration (IC₅₀ values), Fab/IgG titration was performed and IC₅₀was calculated using GraphPad Prism.

For the evaluation of a potential agonistic activity of the HuCAL® IgGs,as determined by the ability of the antibodies to promote proliferationof the engineered BaF/3 cells in the absence of exogenous FGF1, thecells and IgGs were diluted in dilution medium (full-growth mediumwithout FGF1). Seeding of the cells and the addition of the IgGs wasdone at the same day.

Affinity Determination

For K_(D) determinations, monomer fractions of antibody protein wereused (at least 90% monomer content, analyzed by analytical SEC;Superdex75 (Amersham Pharmacia) for Fab, or Tosoh G3000SWXL (TosohBioscience) for IgG, respectively).

(a) Solution Equilibrium Titration (SET) Method for KD DeterminationUsing Sector Imager 6000 (Mesoscale discovery)

Affinity determination in solution was basically performed as describedin the literature (Friguet et al., 1985 J Immunol Methods 77: 305-319).In order to improve the sensitivity and accuracy of the SET method, itwas transferred from classical ELISA to ECL based technology (Haenel etal., 2005 Anal Biochem 339: 182-184). K_(D) determination of HuCAL®_IgGwas performed as described using the following reagents: biotinylatedhFGFR2. was coated at 0.5 μg/ml in PBS overnight at 4° C. on standardMSD plates.

After washing the MSD plate and adding 30 μl/well MSD Read Buffer T withsurfactant, electrochemiluminescence signals were detected using aSector Imager 6000 (MesoScale Discovery, Gaithersburg, Md., USA).

The data was evaluated with XLfit (IDBS) software applying customizedfitting models. For K_(D) determination of Fab molecules the followingfit model was used (according to (Haenel et al., 2005 Anal Biochem 339:182-184):

$y = {B_{\max} - \left( {\frac{B_{\max}}{{2\lbrack{Fab}\rbrack}_{t}}\left( {\lbrack{Fab}\rbrack_{t} + x + K_{D} - \sqrt{\left( {\lbrack{Fab}\rbrack_{t} + x + K_{D}} \right)^{2} - {4{x\lbrack{Fab}\rbrack}^{t}}}} \right)} \right)}$

[Fab]_(t): applied total Fab concentrationx: applied total soluble antigen concentration (binding sites)B_(max): maximal signal of Fab without antigenK_(D): affinity

For K_(D) determination of IgG molecules the following fit model for IgGwas used (modified according to (Piehler et al., 1997 J Immunol Methods201: 189-206)) and a summary of the data is presented in FIG. 1:

$y = {\frac{2B_{\max}}{\lbrack{IgG}\rbrack}\left( {\frac{\lbrack{IgG}\rbrack}{2} - \frac{\left( {\frac{x + \lbrack{IgG}\rbrack + K_{D}}{2} - \sqrt{\frac{\left( {x + \lbrack{IgG}\rbrack + K_{D}} \right)^{2}}{4} - {x\lbrack{IgG}\rbrack}}} \right)^{2}}{2\lbrack{IgG}\rbrack}} \right)}$

[IgG]: applied total IgG concentrationx: applied total soluble antigen concentration (binding sites)B_(max): maximal signal of IgG without antigenK_(D): affinity

Summary of Panning Strategies and Screening

In total, 28 different panning strategies were employed usingrecombinant antigen material as well as cell lines as outlined above.Using the assays described above, panning outputs were screened forspecificity and cross-reactivity and 935 clones were selected forsequencing. Following the screening process described, 15 antibodieswere selected for further characterization as described in the examplesbelow. Of these antibodies, 12433, 12947, 10846 and 12931 were found tobind to the FGFR2 IIIb isoform (Uniprot Accession # P21802-3), 10164,11725, 10220, 11723, and 12944 were found to bind to both human FGFR2IIIb (Uniprot Accession # P21802-3) and IIIc isoforms (Uniprot Accession# P21802-1), and 12425, 12422, 12439, 10918, 10923 and 11722 were foundto bind to both human FGFR2 IIIb (Uniprot Accession # P21802-3) and IIIcisoforms (Uniprot Accession # P21802-1) and also human FGFR4 (UniprotAccession # P22455).

Example 2: Affinity Determination

Affinity of the antibodies to FGFR2 species orthologues and also toFGFR4 was determined using Biacore technology using a Biacore T100instrument (GE Systems) and with CM5 sensor chips.

Briefly, HBS-EP⁺ (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% SurfactantP20) supplemented with 0.5 mg/ml of BSA and 10 μg/ml heparin was used asrunning buffer for all experiments. The immobilization level and analyteinteractions were measured by response unit (RU). Pilot experiments wereperformed to test and confirm the feasibility of the immobilization ofanti-human Fc antibody and the capture of the test antibodies.

For kinetic measurements, experiments were performed in which theantibodies were immobilized to the sensor chip surface and the abilityof the FGFR proteins described above listed in Table 1 to bind in freesolution was determined. Briefly, 50 μg/ml of anti-human Fc antibody atpH 4.75 was immobilized on a CM5 sensor chip through amine coupling atflow rate of 10 μl/minute on all four flow cells to reach 6000 RUs. 3μg/ml of test antibodies were then injected at 10 μl/min for 10 s.Immobilization levels of antibodies were generally kept below 250 RUs.Subsequently, 0.078-100 nM of FGFR receptors, diluted in a 2-foldseries, were injected at a flow rate of 80 μl/min for 7 min over bothreference and test flow cells. Dissociation of the binding was followedfor 10 min using HBS-EP⁺ supplemented with 0.5 mg/ml of BSA and 10 μg/mlheparin as running buffer. After each injection cycle, the chip surfacewas regenerated with 10 mM glycine, pH 2.0 at 60 μl/min for 70 s. Allexperiments were performed at 25° C. and the response data were globallyfitted with a simple 1:1 interaction model (using evaluation softwareversion 1.1 (GE Systems)) to obtain estimates of on rate (k_(a)),off-rate (k_(d)) and affinity (K_(D)).

A summary of the affinity estimates obtained against FGFR2 IIIb speciesorthologues and FGFR4 is presented in Table 4. It was found that all ofthe antibodies bound to the species orthologues of FGFR2 evaluated andthat a subset of the antibodies also bound to human FGFR4. In addition,some of the antibodies (10846, 12433, 12931 and 12947) were unable tobind to the IIIc isoform of human FGFR2 (Uniprot Accession # P21802).All of the antibodies were specific to FGFR2 or bound to both FGFR2 andFGFR4 and none of the antibodies were found to bind appreciably to FGFR1or FGFR3.

TABLE 4 Affinity estimates obtained against FGFR2 IIIb speciesorthologues and FGFR4 Affinity estimate (K_(D)) (M) Mouse Rat CynoAntibody Human FGFR2 FGFR2 FGFR2 Human ID FGFR2 IIIb IIIb IIIb IIIbFGFR4 10164 1.9E−08 3.3E−08 3.5E−08 1.6E−08 N.B. 10220 1.6E−08 2.6E−082.6E−08 2.6E−08 N.B. 10846 1.4E−12 1.5E−08 1.3E−08 2.3E−08 N.B. 109187.7E−10 1.1E−09 1.3E−09 1.3E−09 1.8E−09 10923 3.7E−09 4.0E−09 5.5E−094.5E−09 6.4E−09 11722 1.3E−08 1.8E−08 2.1E−08 1.3E−08 1.1E−08 117231.8E−08 2.8E−08 5.3E−08 1.7E−08 N.B. 11725 2.6E−08 1.3E−07 1.4E−077.3E−09 N.B. 12422 5.1E−09 7.7E−09 9.7E−09 7.4E−09 9.5E−10 12425 1.4E−081.6E−08 2.2E−08 1.2E−08 3.9E−09 12433 5.6E−09 8.0E−09 1.2E−08 1.2E−08N.B. 12439 1.5E−08 1.5E−08 1.6E−08 5.6E−09 4.3E−09 12931 3.4E−09 1.2E−092.9E−09 2.4E−09 N.B. 12944 9.4E−09 6.9E−09 1.1E−08 1.0E−08 N.B. 129472.7E−09 3.1E−09 5.1E−09 2.4E−09 N.B.

Example 3: Assessment of Functional Activity of Anti-FGFR Antibodies

The ability of the purified antibodies to act as either agonists orantagonists of FGFR2 was evaluated using a Baf cell system in which Bafcells were transduced to overexpress FGFR2.

To evaluate potential agonistic properties of the antibodies, Baf-FGFR2cells were washed twice in PBS and re-suspended in dilution medium (RPMIsupplemented with 10% FBS, 2 μg/ml heparin (Sigma, catalog # H3149) and20 μg/ml blasticidin (e.g. Invitrogen, catalog #A11139-03 or Cellgro,catalog #30-100-RB) prior to seeding in 96 well plates (Costar catalog#3904) at 8000 cells/well in 90 μl dilution medium. In each assay, oneplate was designated a “day 0” plate: to each well of these plates, anadditional 10 μl of dilution medium was added, followed 80 μl/well ofCell titer Glo reagent (Promega #G7573). Assay plates were shaken gentlyfor 10 min and the resulting luminescence intensity was measured using aPerkin Elmer EnVision 2101 plate reader. Serial dilutions were preparedfor each antibody as 10× solutions in dilution medium and 10 μl wasadded to the appropriate wells. In addition to the test antibodies, FGF1(Peprotech, catalog #100-17A; final assay concentration 0-30 nM) acommercially available anti-FGFR2 antibody (R&D systems, catalog #MAB6841) and a non-FGFR2 binding antibody were included as controlreagents. Assay plates were incubated for 3 days at 37° C. with 5% CO₂.Following this incubation, the plates were brought to room temperaturefor around 30 min and 80 μl/well of Cell titer Glo reagent (Promega#G7573) was added. Plates were then shaken gently for 10 min and theresulting luminescence intensity was measured using a Perkin ElmerEnVision 2101 plate reader. To determine the effect of the antibodies oncell proliferation, raw luminescence values were averaged acrossreplicates and compared to day 0 controls. 10846, 11725 and 12439 werenot evaluated in these studies but none of the other antibodies testedwere able to substitute for FGF1 and maintain cell growth. A summary ofthe data is presented in FIG. 2 (A)/(B).

The Baf cell system was also used to assess the potential of theantibodies to act as antagonists of FGFR2 receptor signaling. In thesestudies, Baf-FGFR2 cells were plated as described in the agonism studiesabove with the addition of 30 μg/ml FGF1 (Peprotech, catalog #100-17A)to the dilution medium. Serial dilutions were prepared for each antibodyas 4× solutions in dilution medium with 30 μg/ml FGF1 and 25 μl wasadded to the appropriate wells. In addition to the test antibodies, acommercially available anti-FGFR2 antibody (R&D systems, catalog #MAB6841) and a non-FGFR2 binding antibody were included as controlreagents. Assay plates were incubated for 3 days at 37° C. with 5% CO₂.Following this incubation, the plates were brought to room temperaturefor around 30 min and 80 μl/well of Cell titer Glo reagent (Promega#G7573) was added. Plates were then shaken gently for 10 min and theresulting luminescence intensity was measured using a Perkin ElmerEnVision 2101 plate reader. To determine the effect of the antibodies onrelative cell proliferation, raw luminescence values were averagedacross replicates and compared. 10846, 11725 and 12439 were notevaluated in these studies. The effects of the other antibodies testedare presented in FIG. 3(A)/(B) and demonstrate that of the clonesevaluated 10918, 10923, 12931, 12944, 12947 and 12422 inhibited theproliferation of the engineered Baf cells by greater than 50% atconcentrations of less than 100 nM.

Example 4: Preparation of ADCs Preparation of the DM1 Conjugates byOne-Step Process

Antibody 12425 was diafiltered into a reaction buffer (15 mM potassiumphosphate, 2 mM EDTA, pH 7.6) via Tangential Flow Filtration (TFF#1)prior to the start of the conjugation reaction. Subsequently, antibody12425 (5.0 mg/mL) was mixed with DM1 (5.6-fold molar excess relative tothe amount of antibody) and then with SMCC (4.7 fold excess relative tothe amount of antibody). The reaction was performed at 20° C. in 15 mMpotassium phosphate buffer (pH 7.6) containing 2 mM EDTA and 10% DMA forapproximately 16 hours. The reaction was quenched by adding 1 M aceticacid to adjust the pH to 5.50. After pH adjustment, the reaction mixturewas filtered through a multi-layer (0.45/0.22 μm) PVDF filter andpurified and diafiltered into a 20 mM succinate buffer (pH 5.0)containing 8.22% sucrose using Tangential Flow Filtration (TFF#2). Theinstrument parameters for the Tangential Flow Filtration are listed inTable 5 below.

TABLE 5 Instrument parameters for the Tangential Flow Filtration TFFParameter TFF#1 Set Point TFF#2 Set Point Bulk Concentration (Cb-g/L) 2020 TMP (psi) 12-18 12-18 Feed Flow rate (LMH) 324  324  Membrane Load(g/m2) 110-150 110-150 Diavolumes 10 14 Diafiltration Buffer 15 mMpotassium 20 mM Succinate, phosphate, 2 mM 8.22% Sucrose, pH 5.0 EDTA,pH 7.6 Temperature (° C.) RT (20-25) RT (20-25)

Conjugates obtained from the process described above was analyzed by: UVspectroscopy for cytotoxic agent loading (Maytansinoid to AntibodyRatio, MAR); SEC-HPLC for determination of conjugate monomer; andreverse-phase HPLC or hydrophobic shielded phase (Hisep)-HPLC for freemaytansinoid percentage. The data is shown in Table 6.

TABLE 6 Properties of 12425-MCC-DM1 Total Free Sample MAR Monomer (%)Maytansinoid (%) 12425-MCC-DM1 3.6 98.0 1.0

Preparation of DM1 Conjugates by In Situ Process

The conjugates of the present invention can also be prepared by in situprocess according to the following procedures. Antibodies (21 clones)were conjugated to DM1 using the sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) linker.Stock solutions of DM1 and sulfo-SMCC heterobifunctional linker wereprepared in DMA. Sulfo-SMCC and DM1 thiol were mixed together to reactfor 10 minutes at 25° C. in DMA containing 40% v/v of aqueous 50 mMsuccinate buffer, 2 mM EDTA, pH 5.0, at the ratio of DM1 to linker of1.3:1 mole equivalent and a final concentration of DM1 of 1.95 mM. Theantibody was then reacted with an aliquot of the reaction to give a moleequivalent ratio of SMCC to Ab of around 6.5:1 under final conjugationconditions of 2.5 mg/mL of Ab in 50 mM EPPS, pH 8.0 and 10% DMA (v/v).After approximately 18 hours at 25° C., the conjugation reaction mixturewas purified using a SEPHADEX™ G25 column equilibrated with 10 mMsuccinate, 250 mM glycine, 0.5% sucrose, 0.01% Tween 20, pH 5.5.

TABLE 7 Properties of DM1-conjugated antibodies Clone Linker MonomerYield Free drug name excess MAR (%) (%) (%) 10164 6.8 3.5 98 88 <0.510220 7.5 3.55 98 96 0.5 10553 6.4 3.7 100 64 <0.5 10554 6.3 3.5 100 79<0.5 10846 6.3 3.4 99 71 <0.5 10918 6.4 3.6 98 99 0.5 10923 6.2 3.5 10095 0.7 10925 7.9 3.5 99 70 <0.5 11722 9.5 3.5 99 84 3.7 11723 7.3 3.3 9964 <0.5 11725 8.2 3.4 99 74 <0.5 11729 9.5 3.2 93 30 0.7 12422 6.2 4.099 60 <0.5 12425 6.1 3.5 99 70 <0.5 12433 6.1 3.4 99 82 <0.5 12435 7.03.4 99 75 <0.5 12438 7.7 3.4 99 75 <0.5 12439 6.4 3.5 99 99 0.5 129316.4 3.8 99 80 0.5 12944 7.8 3.5 99 38 1.6 12947 6.7 3.8 99 99 <0.5Preparation of ADCs with the SPDB Linker

Antibodies 12422, 12425 and 12433 (8 mg/ml) was modified withN-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB, 5.0, 5.5 and 4.9 foldmolar excess respectively) for 120 minutes at 25° C. in 50 mM potassiumphosphate buffer (pH 7.5) containing 50 mM NaCl, 2 mM EDTA, and 5% DMA.The modified Ab without purification was subsequently conjugated to DM4(1.7 fold molar excess over the unbound linker) at a final modifiedantibody concentration of 4 mg/mL in 50 mM potassium phosphate buffer(pH 7.5) containing 50 mM NaCl, 2 mM EDTA, and 5% DMA for 18 hours at25° C. The conjugation reaction mixture was purified using a SEPHADEX™G25 column equilibrated and eluted with 10 mM succinate, 250 mM glycine,0.5% sucrose, 0.01% Tween 20, pH 5.5.

TABLE 8 Properties of DM4-conjugated antibodies Clone Linker MonomerYield Free drug name excess MAR (%) (%) (%) 12422 5.0 3.6 99 85 0.512425 5.5 3.6 98 >90 <0.5 12433 4.9 4.0 99 75 0.5Preparation of ADCs with the CX1-1 Linker

Antibody 12425 (5.0 mg/mL) was mixed with DM1 (7.15-fold molar excessrelative to the amount of antibody) and then with CX1-1 (5.5-fold excessrelative to the amount of antibody). The reaction was performed at 25°C. in 60 mM EPPS [4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid]buffer (pH 8.5) containing 2 mM EDTA and 5% DMA for approximately 16hours. The reaction mixture was then purified using a SEPHADEX™ G25column equilibrated and eluted in 10 mM succinate, 250 mM glycine, 0.5%sucrose, 0.01% Tween 20, pH 5.5.

TABLE 9 Properties of CX1-1/DM1 conjugated 12425 Clone Linker MonomerYield Free drug name excess MAR (%) (%) (%) 12425 5.5 X 3.4 97 >90 0.1

Example 5: Affinity of ADCs Relative to Parental Antibodies

The affinity of the antibodies to FGFR2 and FGFR4 following conjugationto SMCC-DM1 was determined using Biacore technology using a Biacore T100instrument (GE Systems) and CM5 sensor chips using similar methodologyto that described in example 2 above.

For the antibodies assessed, similar affinity estimates for binding tohuman FGFR2 IIIb were obtained for SMCC-DM1 conjugated antibodiesrelative to parental unconjugated antibodies, suggesting thatconjugation does not appreciably impact antibody binding (Table 10).

TABLE 10 Affinity estimates for unconjugated and SMCC-DM1 conjugatedantibodies Human FGFR2 IIIb affinity (K_(D)) (nM) Unconjugated AntibodyID antiboby Antibody-MCC-DM1 10164 2 2.6 10220 17 7.8 10846 3.5 3.810918 2.2 2.1 10923 2.2 2.3 11722 4.3 8.8 11723 6.6 5.9 11725 3.3 2.912422 5.1 N.D. 12425 2.1 2.3 12433 5.6 4.9 12439 15 N.D. 12931 3.4 N.D.12944 9.4 N.D. 12947 2.7 N.D. N.D. = not determined

In addition, the affinities of several SMCC-DM1 conjugated antibodies toFGFR2 and FGFR4 species orthologues was determined. In these studies, noappreciable differences in affinity were found between the conjugatedand unconjugated antibodies (Table 11).

TABLE 11 Affinities of SMCC-DM1 conjugated antibodies to FGFR2 and FGFR4species orthologues Affinity (K_(D)) (nM) 12433- MCC- 10164- 12425-Protein 12433 DM1 10164 MCC-DM1 12425 MCC-DM1 Human 5.6 4.9 2 2.6 2.12.3 FGFR 2IIIb Mouse 2.2 1.2 10 10 3.2 3.6 FGFR2 IIIb Rat FGFR2 3.7 2.82.8 3.5 3.1 3.5 IIIb Cynomolgus 1.7 1.5 9 3.8 1.6 1.9 monkey FGFR2 IIIbHuman No binding N.D. No binding N.D. 4.0 4.6 FGFR4 Mouse N.D. N.D. N.D.N.D. 3.6 2.2 FGFR4 Rat FGFR4 N.D. N.D. N.D. N.D. 5.2 4.2 Cynomolgus N.D.N.D. N.D. N.D. 2.3 2.1 monkey FGFR4 N.D. = not determined

Example 6: In Vitro Activity ofADCs in SNU16 and Kato-III Cells

Following conjugation to the SMCC-DM1 linker-payload, the ability of theantibody drug conjugates (ADCs) to inhibit the proliferation ofFGFR2-amplified cell lines, SNU16 (ATCC catalog #CRL-5974) and Kato-III(ATCC catalog # HTB-103) was determined. Briefly, cells were cultured ina tissue culture incubator at 37° C. with 5% CO₂ in culture medium asrecommended by the supplier. On the day of the assay, cells were washedtwice with PBS (Lonza, catalog #17516C), prior to being treated with0.25% trypsin-EDTA (Gibco catalog #25300) for 5 min and resuspension inthe recommended culture medium. Cells were then counted and seeded in 96well plates (Costar catalog #3940 or Corning catalog #3340) at densitiesof 2600-3600 cells/well in 100 μl of cell culture medium. A duplicateplate was generated for a day 0 measurement and all plates wereincubated in a tissue culture incubator at 37° C. with 5% CO₂ overnight.Medium only wells were also generated to act as negative controls.Following this incubation, 50 μl/well of Cell titer Glo reagent (Promegacatalog # G7573) was added to the day 0 plates, which were then shakengently for 10 min and the resulting luminescence intensity was measuredusing a Perkin Elmer EnVision 2101 plate reader. Test ADCs were seriallydiluted in duplicate to a 2× stock solution in the appropriate cellculture medium and, following the removal of 50 μl of medium from eachof rest of the assay plates, 50 μl of 2× serially diluted ADCs wereadded (final assay concentration 0.2-50 nM DM1 equivalents) prior toincubation in a tissue culture incubator at 37° C. with 5% CO₂ for 5days. Following this incubation period, relative cell viability wasdetermined via the addition of Cell titer Glo reagent as describedabove. The effect of the ADCs on cell proliferation was calculated usingthe average of the duplicates as follows: % Inhibition=(ADCtreated−untreated)/(untreated−Day 0)*100 The % inhibition data wasfitted to a 4-parameter logistic equation and IC₅₀ values weredetermined. Unlike the IgG control ADC, the tested ADCs were found to bepotent inhibitors of the proliferation of both Kato-III and SNU16 cellswith IC₅₀'s of less than 3 nM (Table 12).

TABLE 12 Antibody potency as MCC-DM1 ADCs Cell proliferation IC50 (nM)Antibody ID Kato-III SNU16 10164 1.6 0.5 10220 0.33 <0.2 10846 0.43 <0.210918 0.61 <0.2 10923 0.45 <0.2 11722 0.59 <0.2 11723 0.91 <0.2 117255.2 5 12422 <3 <3 12425 0.15 <0.2 12433 <3 <3 12439 <3 <3 12931 <3 <312944 <3 <3 12947 <3 <3 IgG control ADC >50 >25

The ability of several anti-FGFR antibodies conjugated via the SPDB-DM4linker-payload was also evaluated. These studies, which were conductedas described above, revealed that the antibodies evaluated were alsopotent inhibitors of cell proliferation as SPDB-DM4 ADCs, suggestingthat their ability to successfully deliver payloads to kill cells is notlimited to MCC-DM1. Data for the SNU16 cell line is summarized in Table13.

TABLE 13 Antibody potency as SPDB-DM4 ADCs Cell proliferation AntibodyID IC50 (nM) 12422 0.07 12425 0.04 12433 0.8 IgG control ADC >25

Example 7: Assessment of In Vivo Activity of Anti-FGFR2/4 ADCs

The antitumor activity of 15 anti-FGFR2 or anti-FGFR2/FGFR4cross-reactive ADCs was evaluated in the gastric, FGFR2 amplified(CN=31), FGFR2 IIIb isoform expressing SNU16 xenograft tumor model. Intwo independent studies, female nude mice were implanted subcutaneouslywith 10×10⁶ cells in a suspension containing 50% phenol red-freematrigel (BD Biosciences) in Hank's balanced salt solution. The totalinjection volume containing cells in suspension was 200 μl. Mice wereenrolled in the first study eight days post implantation with averagetumor volume of 192.9 mm³. After being randomly assigned to one of ninegroups (n=8/group), mice were administered PBS or a single 3 mg/kgintravenous (i.v.) injection of one of the following antibody drugconjugates: 12433-MCC-DM1, 10164-MCC-DM1, 10220-MCC-DM1, 10918-MCC-DM1,10923-MCC-DM1, 11722-MCC-DM1, 12422-MCC-DM1, or 12425-MCC-DM1. Mice wereenrolled in the second study seven days post implantation with averagetumor volume of 223.4 mm³. After being randomly assigned to one of ninegroups (n=8/group), mice were administered PBS or a single 3 mg/kg i.v.injection of one of the following antibody drug conjugates:12433-MCC-DM1, 12947-MCC-DM1, 12931-MCC-DM1, 12944-MCC-DM1,12439-MCC-DM1, 10846-MCC-DM1, 11725-MCC-DM1, or 11723-MCC-DM1.12433-MCC-DM1 was included as a bridge between the two studies tofacilitate direct comparison of antibody drug conjugates in bothstudies. Tumors were calipered twice per week. ADCs evaluated in thesestudies exhibited a broad spectrum of anti-tumor activity 23 to 24 dayspost dose (ranging from 33.2% T/C to 77.8% regression) (FIGS. 4 (A) and(B)).

The antitumor activity of cleavable disulfide linker based SPDB-DM4FGFR2 ADCs was evaluated in the SNU16 xenograft tumor model. In thesestudies, female nude mice were implanted subcutaneously with 10×106cells in a suspension containing 50% phenol red-free matrigel (BDBiosciences) in Hank's balanced salt solution. The total injectionvolume containing cells in suspension was 200 μl. Mice were enrolledonto study 11 days post implantation with average tumor volume of 223mm³. After being randomly assigned to one of five groups (n=7/group),mice were administered PBS (10 ml/kg), a single 15 mg/kg intravenousinjection of 12433-MCC-DM1 or control IgG-MCC-DM1, or a single 5 mg/kgintraveneous injection of 12433-SPDB-DM4 or IgG-SPDB-DM4. Tumors werecalipered twice per week. Neither control ADCs exhibited antitumoractivity in this study (FIG. 4(C)). However, in contrast, both12433-MCC-DM1 and 12433-SPDB-DM4 were highly active against the SNU16xenograft model at the doses administered, suggesting that anti-tumoreffects of anti-FGFR ADCs can be obtained with different linkers.

The pharmacokinetics (PK) of anti-FGFR ADCs was evaluated in SNU16tumor-bearing mice following a single IV dose of 3 mg/kg. PK sampleswere collected at 1 h, 24 h, 72 h, 168 h, and 336 h post dose. In allstudies, serum concentrations of both “total” antibody and “antibodydrug conjugate (ADC)” were measured in all animal species usingvalidated ELISA methods. The “total” fraction refers to the measurementof the antibody with or without the conjugated DM1, whereas the ADCfraction refers to the measurement of DM1 conjugated antibody only (≧1DM1 molecule). In all studies, no appreciable differences were observedbetween clearance of the ADC and total antibody fractions. A summary ofthe PK properties of the ADCs is presented in FIG. 4 (D)-(E). When theADC clearance following a single 3 mg/kg dose in SNU16 tumor bearingmice was compared to the observed anti-tumor effect, it was found thatthe ADCs with the greatest anti-tumor activity (>40% tumor regression)were all cleared at a rate of less than 45 ml/d/kg (FIG. 4 (F)).

Example 8: Assessment of Signaling Activity of Unconjugated andConjugated Antibodies In Vitro Using FGFR2-Amplified Cell Lines

The ability of the unconjugated and SMCC-DM1 conjugated anti-FGFR2antibodies to modulate FGFR signaling in FGFR2-amplified cell lines wasevaluated. In initial studies, the effect of the 12433, 12425 and 10164antibodies in SNU16 (ATCC catalog #CRL-5974) cells was determined.Briefly, cells were seeded in RPMI supplemented with 10% FBS in 12 wellCellBind plates (Costar catalog #3336) and incubated overnight in atissue culture incubator at 37° C. with 5% CO₂. On the day of theexperiment, the cell culture medium was aspirated and replaced witheither test antibodies or the small molecule FGFR inhibitor, BGJ398, alldiluted in RPMI supplemented with 10% FBS at final concentrations of(13-130 nM, antibodies/ADCs; 500 nM, BGJ398). Cells were then incubatedin a tissue culture incubator at 37° C. with 5% CO₂ for 2 h. Followingthis incubation, cells were washed twice with cold PBS (Lonza, catalog#17516C), placed on ice and 300 μl of lysis buffer (CST catalog #9803with PhosphoSTOP Roche catalog #04 906237001) was added. Proteinconcentration was determined by BCA assay (Pierce catalog #23228). 60 μgof protein was then resolved by SDS-PAGE, transferred ontonitrocellulose membranes and probed with antibodies to pFRS2 (CellSignaling Technology, catalog #3861), total FGFR2 (e.g. Santa CruzBiotechnology, catalog # SC-122 or R&D systems catalog #6841) andβ-actin (Bethyl, catalog # A300-485A) or cytokeratin (Dako catalog #M3515). Results are presented in FIG. 5 (A) and it was found that noneof the antibodies, either unconjugated or as MCC-DM1 ADCs were able tomodulate pFRS2 signal at 2 h. Further experiments were conducted withthe 12425 antibody (4.3 nM) and 12425-MCC-DM1 ADC (4.3 nM antibody, 13nM DM1 equivalents) in which additional time points (4, 24 and 72 h)were evaluated in both SNU16 and also Kato-III (ATCC catalog # HTB-103)cells. Results are presented in FIG. 5 (B) (SNU16) and 5 (C) (Kato-III).No effects of either the naked antibody or ADC were observed up to 48 hin SNU16 cells or up to 72 h in Kato-III cells. A reduction in bothpFRS2 and tFGFR2 signal was observed at 72 h in SNU16 cells relative tocytokeratin following 12425-MCC-DM1 treatment. As described in example 9below, 12425-MCC-DM1 is a potent inhibitor of SNU16 proliferation at the72 h timepoint and this decrease likely reflects heterogeneity of FGFR2expression in the SNU16 cell population.

Example 9: Assessment of ADC and Unconjugated Antibodies on CellularProliferation

The ability of the unconjugated and SMCC-DM1 conjugated anti-FGFRantibodies to inhibit the proliferation of a panel of FGFR2-amplified,over-expressing and null cell lines was evaluated. In these studies,cells were cultured in a tissue culture incubator at 37° C. with 5% CO₂in culture medium as recommended by the supplier. On the day of theassay, cells were washed twice with PBS (Lonza, catalog #17516C), priorto being treated with 0.25% trypsin-EDTA (Gibco catalog #25300) for 5min and resuspension in the recommended culture medium. Cells were thencounted and seeded in either 96 or 384 well plates (e.g. Costar catalog#3940, Corning catalog #3340, Costar catalog #3707) at densities of800-3600 cells/well in 55-100 μl of cell culture medium. A duplicateplate was generated for a day 0 measurement and all plates wereincubated in a tissue culture incubator at 37° C. with 5% CO₂ overnight.Medium only wells were also generated to act as negative controls.Following this incubation, 30-50 μl/well of Cell titer Glo reagent(Promega catalog # G7573) was added to the day 0 plates, which were thenshaken gently for 10 min and the resulting luminescence intensity wasmeasured using a Perkin Elmer EnVision 2101 plate reader. Test ADCs wereserially diluted in duplicate to either a 2× or 10× stock solution inthe appropriate cell culture medium. For assays in which a 2×ADC stocksolution was used, half of the assay media was removed and replaced withan equal volume of the 2× serially diluted ADCs. Where a 10×ADC stocksolution was employed, this was diluted 1:10 into the assay plates (e.g.5 μl to 55 μl) prior to incubation in a tissue culture incubator at 37°C. with 5% CO₂ for 5 or 6 days. Final assay concentration of the ADCsranged from 0.005-100 nM DM1 equivalents. Following this incubationperiod, relative cell viability was determined via the addition of Celltiter Glo reagent as described above. The effect of the ADCs on cellproliferation was calculated using the average of the duplicates asfollows:

% Inhibition=(ADC treated−untreated)/(untreated−Day 0)*100

The % inhibition data was fitted to a 4-parameter logistic equation andIC₅₀ values were determined. Representative data for 12425-MCC-DM1 ispresented in FIG. 6 (A). This ADC was found to be a potent inhibitor ofcell proliferation in both the SNU16 (FIG. 6 (A)) and Kato-IIIFGFR2-amplified cell lines (FIG. 6 (B)). These cell lines were bothconfirmed to be sensitive to the maytansinoid payload (L-Me-DM1; freeDM1 in FIG. 6 (A)-(C)) but not to a non-targeting ADC (directed tochicken lysozyme) conjugated via SMCC-DM1 at a similar maytansinoidantibody ratio. 12425-MCC-DM1 was also not active against a gastriccancer cell line, NUGC3, that is devoid of appreciable FGFR2 expressionbut that is sensitive to the maytansinoid payload (FIG. 6 (C)). Inaddition, the unconjugated antibody, 12425, was found to lackanti-proliferative activity in both SNU16 (FIG. 6 (D)) and Kato-IIIcells (data not shown). A summary of the anti-proliferative activity ofseveral anti-FGFR-MCC-DM1 ADCs in FGFR2 amplified cell lines, includingSNU16, Kato-III, SUM-52, MFM223 and H716 cells (Kunii et al., 2008Cancer Res 68: 2340-2348; Turner et al., 2010 29: 2013-2023; Mathur etal., 2010. Proceedings of the 101^(st) Annual Meeting of the AmericanAssociation for Cancer Research, poster #284) is shown in Table 14. Inaddition, none of the ADCs were found to be active against a panel oftumor cell lines, including AZ521, CAL-51, KYSE-150, TE-6, SNU-1041, TT,CHL-1, G401 and HEC59.

TABLE 14 Anti-proliferative activity of several anti-FGFR-MCC-DM1 ADCsin FGFR2 gene amplified & non-amplified cell lines In vitro potency(IC₅₀, nM free DM1 equivalents) FGFR2 12433- 10164- 12425- IgG- CellLine Lineage amplification? DM1 DM1 DM1 DM1 SNU16 Gastric Yes (IIIb)0.08 0.04 0.03 11 Kato-III Gastric Yes (IIIb) 0.28 0.32 0.34 30 SUM-52Breast Yes (IIIb) 0.29 0.14 0.17 >30 MFM223 Breast Yes (IIIb) 24 3.51 >30 H716 Colon Yes (IIIb) >30 1.7 0.1-0.7 >30 NUGC3 Gastric No(IIIb) >30 >30 >30 >30

Example 10: In Vivo PK-PD of FGFR ADCs

Studies were conducted to assess the ability of 12425-MCC-DM1 tomodulate pharmacodynamic markers in vivo. The goal of these studies wasto evaluate the relationship between FGFR2 or FGFR4 expression and G2/Mcell cycle arrest. Accumulation of pHH3 positive nuclei, as assessed byimmunohistochemistry, was used as a marker of G2/M arrest.

A rabbit polyclonal antibody produced by immunizing animals with asynthetic phosphopeptide corresponding to residues surrounding Ser10 ofhuman histone H3 was obtained from Cell Signaling Technology (Danvers,Mass.). Briefly, the IHC protocol included heat and standard exposure toVentana Cell Conditioning #1 antigen retrieval reagent for approximately48 minutes at a temperature ranging from 93° C. to 95° C. The primaryantibody was diluted to 1:100 and incubated for 60 min at roomtemperature. Subsequently, incubation with Ventana OmniMap pre-dilutedHRP-conjugated anti-rabbit antibody (Cat #760-4311) was performed for 4min.

To assess PD in the FGFR2 amplified SNU16 xenograft model, female nudemice were implanted subcutaneously with 10×10⁶ cells in a suspensioncontaining 50% phenol red-free matrigel (BD Biosciences) in Hank'sbalanced salt solution. The total injection volume containing cells insuspension was 200 μl. Six mice were randomly assigned to receive ani.v. dose of either 12425-MCC-DM1 (10 mg/kg) or PBS (10 ml/kg) oncetumors reached between 300 and 500 mm³ (n=3/group). Consistent with theexpected mechanism of action of the maytansinoid payload, 12425-MCC-DM1yielded a marked, time-dependent increase in nuclear pHH3 positively 24h post dose relative to PBS treated controls (representative imagesshown in FIG. 7 (A)). Time dependent changes in cleaved caspase 3 werealso evaluated. In these studies, a rabbit polyclonal antibody producedby immunizing animals with a synthetic peptide corresponding toamino-terminal residues adjacent to (Asp 175) in human caspase-3 wasobtained from Cell Signaling Technology (Danvers, Mass.). The IHCprotocol included No Heat and Standard exposure to Ventana CellConditioning #1 antigen retrieval reagent for approximately 48 minutesat a temperature ranging from 93° C. to 95° C. The primary antibody wasdiluted to 1:300 and incubated for 60 minutes at room temperature.Subsequently, incubation with Ventana OmniMap prediluted HRP-conjugatedanti-rabbit antibody (Cat #760-4311) was performed for 4 minutes.Similar to pHH3, time dependent changes in cleaved caspase 3 were alsoobserved (FIG. 7(A)). Similar data was also obtained for otheranti-FGFR2 ADCs e.g. 12433-MCC-DM1.

To assess ADC specificity, PD was evaluated in the FGFR2/FGFR4 negativeNUGC3 xenograft model. In these studies, female nude mice were injectedsubcutaneously with 1×10⁶ cells in a suspension containing 50% phenolred-free matrigel (BD Biosciences) in Hank's balanced salt solution. Thetotal injection volume containing cells in suspension was 200 μl. Ninemice were randomly assigned to receive a single intravenous 15 mg/kgdose of 12425-MCC-DM1, control IgG-MCC-DM1 or PBS (10 ml/kg) once tumorsreached between 300 and 500 mm³ (n=3/group). 12425-MCC-DM1 failed tomodulate pHH3 levels relative to a 15 mg/kg i.v. dose of controlIgG-MCC-DM1 in FGFR2 and FGFR4 negative NUGC3 xenografts (representativeimages shown in FIG. 7(B)). Similar data was also obtained for otheranti-FGFR2 ADCs e.g. 12433-MCC-DM1.

Taken together, these data demonstrate that 12425-MCC-DM1 is capable ofeliciting robust in vivo cellular PD effects that are dependent uponFGFR2 or FGFR4 expression and consistent with the mechanism of action ofthe maytansinoid payload.

Example 11: In Vivo Efficacy of Anti-FGFR ADCs

The anti-tumor activity of anti-FGFR2 and/or anti-FGFR2/4 ADCs wasevaluated in several tumor xenograft models.

The antitumor activity of three anti-FGFR2 or anti-FGFR2/FGFR4dual-targeting ADCs was evaluated in the gastric, FGFR2 amplified (copynumber=31, SNP6.0), FGFR2 IIIc isoform expressing NCI-H716 colorectalxenograft tumor model. Female nude mice were implanted subcutaneouslywith 5×10⁶ cells containing 50% phenol red-free matrigel (BDBiosciences) in Hank's balanced salt solution. The total injectionvolume containing cells in suspension was 200 μl.

Mice were enrolled in the study four days post implantation with averagetumor volume of 171.7 mm³ (FIG. 8 (A)). After being randomly assigned toone of eight groups (n=6/group), mice were administered PBS (10 ml/kg),a single i.v. dose of control IgG-MCC-DM1 (15 mg/kg), 12422-MCC-DM1 (5or 15 mg/kg), 12425-MCC-DM1 (5 or 15 mg/kg), or 10164-MCC-DM1 (5 or 15mg/kg). Tumors were calipered twice per week. The control IgG-MCC-DM1was not active at 15 mg/kg. 10164-MCC-DM1 was not active at 5 mg/kg.10164-MCC-DM1 (15 mg/kg), 12422-MCC-DM1 (5 and 15 mg/kg) and12425-MCC-DM1 (5 and 15 mg/kg) had exhibited similar activity through 14d post dose. 12425-MCC-DM1 (5 mg/kg and 15 mg/kg) and 10164-MCC-DM1 (15mg/kg only) yielded the most durable response through 18 d post dose(23, 21, and 31% T/C, respectively).

The dose response antitumor activity of three anti-FGFR2 oranti-FGFR2/FGFR4 ADCs was also evaluated in the gastric, FGFR2amplified, FGFR2 IIIb isoform expressing MFM223 ER/PR/HER2 negativebreast xenograft tumor model. The MFM223 model was established as afragment based model. One day before fragment implantation, female nudemice were implanted subcutaneously with 0.72 mg, 60 day sustainedrelease 17β-estradiol pellet (Innovative Research of America) tomaintain serum estrogen levels. One day after 17β-estradiol pelletimplantation, xenografts harvested from donor mice were cut into 3 by 3mm³ fragments subcutaneously implanted into recipient nude female mice.Mice were enrolled in the study 21 days post implantation with anaverage tumor volume of 208.4 mm³ (FIG. 8 (B)). After being randomlyassigned to one of five groups (n=8/group), mice were administered PBS(10 ml/kg) or a single 10 mg/kg i.v. dose of control IgG-MCC-DM1,12433-MCC-DM1, 10164-MCC-DM1, or 12425-MCC-DM1. Tumors were caliperedtwice per week. IgG-MCC-DM1 was not active in this model. A single 10mg/kg i.v. dose of 12433-MCC-DM1, 12425-MCC-DM1, and 10164-DM1 yielded37, 24, and 7% T/C 18 d post administration.

The activity of 12425-MCC-DM1 as a single agent was also evaluated in anFGFR2-amplified patient-derived primary gastric tumor xenograft model,CHGA-010 (FGFR2 copy number=48, (SNP6.0)). In these studies, femalenu/nu athymic mice were implanted subcutaneously with 3×3×3 mm tumorfragments containing 50% phenol red-free matrigel (BD Biosciences) inDMEM. The tumor take rate was >50% and reached approximately 250 mm3 at4 weeks post implantation. Following a single dose of 10 mg/kg IV12425-MCC-DM1, tumor stasis as achieved through approximately 16 to 20days post-dose (FIG. 8 (C)). Similar single dose data was also obtainedfor the FGFR2 specific ADC, 12433-MCC-DM1 (data not shown). In addition,a group receiving 10 mg/kg q3w*2 IV maintained roughly tumor stasisthrough 37 d post initial dose while tumors in the group receiving asingle 10 mg/kg dose averaged 1588 mm³ by 37 d post initial dose. Thesedata demonstrate that the CHGA010 xenografts are able to respond to asecond dose of 12425-MCC-DM1.

Example 12: Improved Anti-Tumor Activity of FGFR ADC with Small MoleculeFGFR Inhibitor, BGJ398

Anti-tumor activity of FGFR2/4 antibody drug conjugate 12425-MCC-DM1alone and in combination with the FGFR small molecule tyrosine kinaseinhibitor, BGJ398 (Guagnano et al., 2001 J Med Chem 54: 7066-7083) wasevaluated in the FGFR2-amplified CHGA119 patient-derived primary gastrictumor xenograft model (FGFR2 copy number=22 (SNP6.0)). In these studies,CHGA119 tumor fragments in size of 2 mm×2 mm×2 mm were implantedsubcutaneously (s.c.) into female nu/nu athymic mice. Forty days afterimplantation, mice carrying CHGA119 tumors (n=8, average 251 mm³; range:104-382 mm³) were treated with vehicle (50% PEG300 in aceticacid/acetate buffer) orally via gavage (pH4.6, 10 ml/kg, p.o., qd),control IgG-3207-DM1 (10 mg/kg, i.v., q2 wk), 12425-MCC-DM1 (10 mg/kg,i.v., q2 wk), NVP-BGJ398-AZ-3 (10 mg/kg, p.o., qd), or the combinationof 12425-MCC-DM1 and NVP-BGJ398-AZ-3, respectively. Tumors werecalipered twice a week. 12425-MCC-DM1 and BGJ398 each as single agentresulted in tumor stasis or partial response (T/C=4% or 33%,respectively, p<0.05), while the treatment in combination of12425-MCC-DM1 and BGJ398 induced near complete tumor regression after 5weeks of treatment (Regression=−94%, p<0.05) (see FIG. 9 and Table 15).Less than 10% body weight loss was observed following either the singleagent or combination treatments (Table 15). These data suggest thatcombining FGFR ADCS with with small molecule inhibitors of FGFRsignaling (e.g. BGJ398, TKI258, ponatinib, AZD4547) can improveanti-tumor responses.

TABLE 15 Effect of anti-FGFR agents on tumor and host parameters inCHGA119-tumor bearing mice Tumor response Mean Host response tumor Mean% body volume body weight change weight change Regression (mm³ ± change(g ± (mean ± Survival Treatment T/C (%) (%) SEM) SEM) SEM)(survivors/total) Vehicle 100 — 1168 ± 204 −1.3 ± 0.3 −5.7 ± 1.4 8/8IgG-MCC- 111 — 1301 ± 243 −1.7 ± 0.6 −7.6 ± 2.7 8/8 DM1 12425-MCC- 4 — 43 ± 35   0.6 ± 0.2   2.5 ± 1.0 8/8 DM1 BGJ398 33 —  386 ± 169 −0.8 ±0.7 −3.7 ± 3   8/8 12425-MCC- — 94 −223 ± 32  −3.7 ± 3.8 −4.3 ± 3.8 7/8DM1 + BGJ398

Example 13: Effect of Dose Fractionation on In Vivo Efficacy of FGFRADC

To better understand the role 12425-MCC-DM1 C_(max) (peak exposure) orC_(avg) (average exposure over a dosing interval) play in its antitumoractivity, a time-dose fractionation experiment was conducted using theSNU16 xenograft model. A range of dose schedule intervals and doselevels yielding a predicted fixed total exposure over a fixed treatmentduration were studied. At certain dose levels, FGFR2 ADCs exhibitnonlinear PK due to TMDD (see example 19). Therefore, in order tomaintain a fixed total exposure across all dose schedule intervals anddose levels, PK modeling predicted that a larger total dose of12425-MCC-DM1 would be required when administered at a lower, morefrequent dose schedule as TMDD would be more pronounced at lower doses.PK modeling predicted that 12425-MCC-DM1 administered at 3 mg/kg q3w*2,2.5 mg/kg q2w*3, 1.5 mg/kg qw*6, and 0.7 mg/kg q3d*14 would achievesimilar total exposure.

Female nude mice were implanted subcutaneously with 10×10⁶ cells in asuspension containing 50% phenol red-free matrigel (BD Biosciences) inHank's balanced salt solution. The total injection volume containingcells in suspension was 200 μl. Mice were enrolled in the first studyten days post implantation with average tumor volume of 181.7 mm³.

To assess PK parameters, serum was collected via tail nick orretro-orbital bleeds and analyzed via ELISA. The total antibody PK assaymeasures total antibody concentration, with/without DM1 by colorimetricELISA. Plates are coated with anti-human IgG (Fc specific), anddetection is with donkey anti-human IgG-HRP before being read on anappropriate plate-reader. The conjugate PK assay measures antibody thatis bound to at least 1 DM1 molecule by colorimetric ELISA. In thisformat, plates are coated with anti-DM1 antibody, and detected withdonkey anti-human IgG-HRP. Serum was collected at the following timepoints post initial dose: 3 mg/kg q3w*2 (2, 6, 24, 48, 96, 168, 240,336, 503 h); 2.5 mg/kg q2w*3 (2, 6, 24, 48, 96, 168, 240, 335, 503 h);1.5 mg/kg qw*6 (2, 6, 24, 48, 96, 167, 335, 503, 671, 839 h); 0.7 mg/kgq3d*14 (2, 6, 24, 48, 71, 143, 215, 287, 359, 431, 503, 575, 647, 719,791, 863, 935 h).

After being randomly assigned to one of five groups (n=7/group), micewere administered PBS (10 ml/kg) or 12425-MCC-DM1 administered i.v. at 3mg/kg q3w*2, 2.5 mg/kg q2w*3, 1.5 mg/kg qw*6, or 0.7 mg/kg q3d*14. Asanticipated, C_(max) varied across treatment regimens while C_(avg) wasstatic (Table 16). All treatment regimens were active and yieldedsimilar tumor growth inhibition suggesting that average exposure of12425-MCC-DM1 is the primary driver of antitumor activity (FIG. 10).These findings indicate that a variety of dose schedules could beemployed in the clinic without compromising efficacy.

TABLE 16 PK parameters following administration of different doses andschedules of 12425-MCC-DM1 to SNU16 tumor-bearing mice Cmax*,Cavg_(Tau), Total ug/ml ug/ml dose, Intact Intact Dosing Regimen mgTotal† ADC‡ Total† ADC‡ Tau, h 3 mg/kg, iv, 6 24.5 25.1 2.19 2.29 503q3wx2 2.5 mg/kg, iv, 7.5 21.2 21.6 2.59 2.30 335 q2wx3 1.5 mg/kg, iv, 911.0 11.0 2.56 2.50 167 qwx6 0.72 mg/kg, iv, 10 4.0 4.2 1.94 1.63 72q3dx14

Example 14: Evaluation of ADCC Activity In Vitro and In Vivo

The ability of the unconjugated anti-FGFR2 antibodies (12433, 10164,12425, N297A_12425 (an ADCC-depeleted variant (Bolt et al., 2003 Eur JImmunol 23: 403-411))) to mediate antibody dependent cellularcytotoxicity was determined versus Kato-III cells (target cells; ATCCcatalog # HTB-103) in co-incubation with NK3.3 cells (killer cells oreffector cells; kindly provided by Jacky Kombluth from San LouisUniversity). In brief, Kato-III cells were stained with Calceinacetoxy-methyl ester (Calcein-AM; Sigma-Aldrich catalog #17783-5MG),washed twice, pipetted into a 96-well microtiterplate (96 well,U-bottomed, clear plastic; Corning Costar, catalog #650 160) at aconcentration of 5000 cells per well and pre-incubated for 10 min with aserial dilution of the above mentioned antibodies and proteins (from50,000 to 0.003 μg per ml) before adding the effector cells. In order tocalculate the antibody specific lysis of the target cells, a parallelincubation of target cells only without antibody or effector cellsserved as a baseline and negative control, whereas the positive controlor maximal lysis or hundred percent specific lysis was determined bylysis of target cells only with a 1% Triton-X 100 solution. Following aco-incubation of target and effector cells in a ratio of one to five,the microtiterplate was centrifuged and an aliquot of the supematantfluid was transferred to another microtiterplate (96 well,flat-bottomed, black with clear bottom; Corning Costar, catalog #3904)and the concentration of free Calcein in solution was determined with afluorescence counter (Victor 3 multilabel counter, Perkin Elmer).Results are presented in FIG. 11 (A) and it was observed that all testedantibodies mediated ADCC to a varying extent. The N297A_12425, carryingno glycosylation in the Fc-part of the antibody and thus lacking thebinding to the CD16a receptor of the effector cells, did not mediate anyADCC and served as a specific negative control. The constant andprotein-concentration independent killing of target cells is due to aunspecific background, natural killing activity of the NK3.3 cellsagainst the Kato-III cells. The antibody 12433 mediated the highestspecific lysis of target cells with about sixty percent (signal deducedthe background lysis of Kato-III cells by NK3.3 cells), whereas theantibodies 12425 and 10164 reached a specific lysis of about fifty-fivepercent (signal deduced the background lysis of Kato-III cells by NK3.3cells). The potency (the concentration at the half maximal killing ofeach dose response curve; effective concentration 50, EC₅₀) of all threemolecules was similar. In a similar series of experiments, the abilityof the naked antibody 12425 to induce ADCC was compared 12425-MCC-DM1.No differences between the naked 12425 and 12425-MCC-DM1 were observed(data not shown) suggesting that DM1 conjugation does not appreciablyimpair the ability of the naked antibody to induce ADCC.

In additional studies, the ability of the unconjugated anti-FGFR2antibodies to bind the complement factor C1q was evaluated. The bindingof the complement factor C1q is the initial step leading to complementdependent cytotoxicity (CDC) and cell lysis in vivo. In these studiesthe surface of the wells of 96-well microtiterplates (Nunc, Maxisorp,catalog #439454) were coated with serial dilutions of the antibodies12433, 12425, N297A_12425 and 10164 over night at 4° C. in the dark (allin a concentration range from 25 to 0.02 μg/ml). The commerciallyavailable antibody, Rituximab served as a positive control. In order tomonitor the amount of bound antibody analyte on the microtiterplate,this procedure was performed twice per plate utilizing the seconddilution (in triplicate) for determining the coating efficacy of theanalyte to the plate. C1q binding was quantified by adding a constantconcentration of C1q (Sigma; Complement component C1q catalog # C1740-1mg) and detected using a polyclonal goat-anti-human C1q antibodyconjugated to Horse Radish Peroxidase HRP (AbD Serotec; Sheep Anti-HumanC1q:HRP; Catalogue number 2221-5004P). The control of the coatingefficiency was determined in the second dilution series with aperoxidase-conjugated goat-anti-human IgG antibody fragment (JacksonImmunoResearch; catalog #109-036-003; AffiniPure F(ab′2) fragment GoatAnti-Human IgG (H+L)). Both were visualized using the TMB substrate (TMBPeroxidase EIA Substrate Kit; Bio-Rad; catalog #172-1067) withsubsequent measurement of optical density at 450 nm with a UV-VisSpectroscope (Molecular Devices; SpectramaxPro 340). Results arepresented in FIG. 11 (B). The maximal amount of C1q protein bound to thetested antibodies was the same for all analytes. The amount of antibodythat was bound to the microtiterplate was the same for all analytesexcept for the 10164 antibody, which reached only around 50% of theother molecules. The potency (the concentration at the half maximalbinding of each dose response curve; effective concentration 50, EC₅₀)of 12433, 10164 and Rituximab was about the same. The EC₅₀ of the 12425antibody was a factor of 3 higher and the EC₅₀ of the N297A_12425 was afactor of about 8 higher. Given the result that 10164 did not coatequally well to the microtiterplate as the other antibodies did, theobserved result in C1q binding implicates an approximate double capacityof 10164 in binding to the C1q protein.

Further to the assessment of C1q binding, the ability of unconjugatedanti-FGFR2 antibodies 12433, 10164 12425 and N297A_12425 to triggercomplement dependent cytotoxicity (CDC) was evaluated. Kato-III cells(target cells; ATCC catalogues number HTB-103) were equally distributedinto the wells of a 96-well microtiterplate (Costar; sterile, white,96-well flat bottomed cell culture plates, catalog #3610) at aconcentration of 10000 cells per well and pre-incubated for 10 min witha serial dilution of the above mentioned antibodies before adding theeffector reagent. The effector reagent used in this study was rabbitcomplement (PelFreez; catalog #31060-1), which was pipetted onto theKato-III cells at a final dilution of one to six. Following a two hourincubation in a humidified cell culture incubator at 37° C., themicrotiterplate was centrifuged, supernatant was discarded and the cellpellet was dissolved in reconstituted CellTiterGlo (Promega;CellTiterGlo Luminescence Kit, catalog # G7572). Luminescence wasquantified in a multilabel reader (Perkin Elmer; Victor 3). Results arepresented in FIG. 11 (C) for all tested antibodies. No evidence of CDCwas observed for the tested antibodies (FIG. 11 (C)) or additionalcontrols (commercially available antibodies Erbitux & Herceptin, datanot shown).

To assess the role of ADCC in 12425-MCC-DM1's in vivo activity, femalenude mice were implanted subcutaneously with 10×10⁶ cells in asuspension containing 50% phenol red-free matrigel (BD Biosciences) inHank's balanced salt solution. The total injection volume containingcells in suspension was 200 μl. Mice were enrolled in the study sevendays post implantation with average tumor volume of 201.4 mm³. Afterbeing randomly assigned to one of five groups (n=6/group), mice receiveda 10 mg/kg i.v. dose of naked control IgG, naked N297A_12425, naked12425, N297A_12425-MCC-DM1, or 12425-MCC-DM1. Tumors were caliperedtwice per week (FIG. 11 (D)). The naked, ADCC-depleted 12425 variant(naked N297A_12425) exhibited less antitumor activity than the ADCCcompetent parental 12425 (61 and 13% T/C, respectively) at 25 d postdose. These data suggest that the parental 12425's effector cellfunction may play a role in the activity of the naked antibody's in vivoantitumor activity. The ADCC competent 12425-MCC-DM1 and ADCC depletedN297A_12425-MCC-DM1 exhibited similar anti-tumor activity at 25 d postdose (45 and 32% regression, respectively). These data suggest thatwhile ADCC activity is expendable, that the maytansinoid payload is bothrequired and sufficient for 12425-MCC-DM1's robust in vivo antitumoractivity.

Example 15: Evaluation of Anti-FGFR Antibodies and ADCs to ModulateSignaling in FGFR4-Overexpressing Cell Lines

The ability of the unconjugated and conjugated anti-FGFR antibodies tomodulate FGFR signaling in FGFR4-overexpressing cell lines wasevaluated. In initial studies, the effect of the 10164 (FGFR2 specific),12433 (FGFR2 specific), 12425 (FGFR2/4 cross-reactive) and associatedMCC-DM1 conjugates in MDA-MB453 (ATCC catalog # HTB-131) cells wasdetermined. Briefly, cells were seeded in RMPI supplemented with 10% FBSin CellBind 12 well plates (Costar, catalog #3336) and incubatedovernight in a tissue culture incubator at 37° C. with 5% CO₂. On theday of the experiment, the cell culture medium was aspirated andreplaced with either test antibodies or the small molecule FGFRinhibitor, BGJ398, all diluted in RPMI supplemented with 10% FBS atfinal concentrations of (13-130 nM, antibodies/ADCs; 500 nM, BGJ398).Cells were then incubated in a tissue culture incubator at 37° C. with5% CO₂ for 2 h. Following this incubation, cells were washed twice withcold PBS (Lonza, catalog #17516C), placed on ice and 300 μl of lysisbuffer (Cell Signaling Technology, catalog #9803 with PhosphoSTOP(Roche, catalog #04 906237001) was added. Protein concentration wasdetermined by BCA assay (Pierce, catalog #23228). Protein concentrationwas determined by BCA assay (Pierce). 60 μg of protein was then resolvedby SDS-PAGE, transferred onto nitrocellulose membranes and probed withantibodies to pFRS2 (Cell Signaling Technology, catalog #3864), totalFGFR4 (R&D Systems, catalog #8652) and β-actin (Bethyl, catalog #A300-485A) or cytokeratin (Dako, catalog # M3515). Results are presentedin FIG. 12 (A) and it was found that none of the antibodies, eitherunconjugated or as MCC-DM1 ADCs were able to modulate pFRS2 signal at 2h. Further experiments were conducted with the 12425 antibody 4.3 nM and12425-MCC-DM1 ADC (4.3 nM antibody, 13 nM DM1 equivalents) in whichadditional time points (4, 24 and 72 h) were evaluated. Results arepresented in FIG. 12 (B). Similar data was also obtained in the RH4rhabdomyosarcoma cell line.

Example 16: Evaluation of Anti-Proliferative Effect of Anti-FGFRAntibodies and ADCs in FGFR4-Overexpressing Cell Lines

The ability of the unconjugated and SMCC-DM1 conjugated anti-FGFRantibodies to inhibit the proliferation of a panel of FGFR4over-expressing cell lines was evaluated using a similar methodology asemployed in example 9 described above.

Consistent with it's cross-reactivity to FGFR4, it was found that12425-MCC-DM1 was a potent inhibitor of the growth of MDA-MB453 cells,which over-express a constitutively active mutant form of FGFR4 (Roidlet al., 2010, Oncogene, 29, 1543-1552). FGFR4 expression was confirmedby FACS analysis using an anti-FGFR4 antibody (Biolegend catalog#324306; FIG. 13 (A)). 12425 demonstrated similar binding properties tothe Biolegend antibody not only in MDA-MB453, but also in otherFGFR4-overexpressing cell lines (data not shown). This effect wasspecific to the ADC, as the unconjugated 12425 antibody was inactive(FIG. 13 (A)). Furthermore, no activity was observed followingadministration of an FGFR2 specific antibody (10164) or ADC(10164-MCC-DM1), suggesting that the effect is driven by FGFR4expression (FIG. 13 (A)). This is consistent with the lack of detectableFGFR2 expression on these cells as assessed by FACs (data not shown). Asimilar effect of 12425-MCC-DM1 and 12425 was also observed in the FGFR4overexpressing rhabdomyosarcoma cell lines, RH4 (FIG. 13 (B)) and JR(FIG. 13 (C)). When extended to additional cell lines, activity of12425-MCC-DM1 was observed in a subset of FGFR4 overexpressing breastand rhabdomyosarcoma cell lines (FIG. 13 (D)).

Example 17: In Vivo PK-PD of FGFR ADCs and Unconjugated Antibodies inFGFR4-Overexpressing Tumors

To assess PD modulation in the FGFR4 positive MDA-MB-453 xenograftmodel, female NSG mice were utilized. One day before cell implantation,female NSG mice were implanted subcutaneously with a 0.36 mg, 90 daysustained release 17β-estradiol pellet (Innovative Research of America).One day after 17β-estradiol pellet implantation, 5×10⁶ cells wereinjected subcutaneously in a suspension containing 50% phenol red-freematrigel (BD Biosciences) in Hank's balanced salt solution. The totalinjection volume containing cells in suspension was 200 μl. Six micewere randomly assigned to receive a single intravenous 15 mg/kg dose ofeither 12425-MCC-DM1 or control IgG-MCC-DM1 once tumors reached between300 and 500 mm³ (n=3/group). 12425-MCC-DM1 yielded a marked increase innuclear pHH3 positively 96 h post dose relative to control micereceiving IgG-MCC-DM1 (representative images shown in FIG. 14 (A)).

To assess the time course of PD modulation in the FGFR4 positiveMDA-MB-453 xenograft model, female NSG mice were utilized. One daybefore cell implantation, female NSG mice were implanted subcutaneouslywith a 0.36 mg, 90 day sustained release 17β-estradiol pellet(Innovative Research of America). One day after 17β-estradiol pelletimplantation, 5×10⁶ cells were injected subcutaneously in a suspensioncontaining 50% phenol red-free matrigel (BD Biosciences) in Hank'sbalanced salt solution. The total injection volume containing cells insuspension was 200 μl. Mice were randomly assigned to receive a singleintravenous 15 mg/kg dose of either 12425-MCC-DM1 (three mice per groupwere collected 24, 48, 72, 96, and 168 hours post treatmentadministration) or PBS (10 ml/kg). Tumor volumes ranged between 300 and500 mm³ at randomization. Consistent with the expected mechanism ofaction of the maytansinoid payload, 12425-MCC-DM1 yielded a marked,time-dependent increase in nuclear pHH3 positivity that peaked around72-96 h h post dose relative to PBS treated controls. A peak in cleavedcaspase 3 immunoreactivity occurred around 96 and 168 h post dose.Representative images shown in FIG. 14 (B).

A portion of alveolar rhabodmyosarcomas, including the RH4 cell line,harbor Pax 3/7-FOXO1A translocations which result in elevated FGFR4 mRNAand protein expression. To assess PD modulation in the RH4 xenograftmodel, female nude mice were implanted subcutaneously with 10×10⁶ cellsin a suspension containing 50% phenol red-free matrigel (BD Biosciences)in Hank's balanced salt solution. The total injection volume containingcells in suspension was 200 μl. Six mice were randomly assigned toreceive a single intraveneous 15 mg/kg dose of either 12425-MCC-DM1,control IgG-MCC-DM1, or PBS (10 ml/kg) once tumors reached between 500and 700 mm³ (n=3/groups). 12425-MCC-DM1 yielded a marked increase innuclear pHH3 positivity 96 h post dose relative to control micereceiving IgG-MCC-DM1 (representative images shown in FIG. 14 (C)).

Example 18: In Vivo Efficacy of Anti-FGFR ADCs in FGFR4-OverexpressingTumors

The dose response antitumor activity of 12425-MCC-DM1 was evaluated inthe FGFR4 positive MDA-MB-453 breast cancer xenograft model. One daybefore cell implantation, female NSG mice were implanted subcutaneouslywith a 0.36 mg, 90 day sustained release 17β-estradiol pellet(Innovative Research of America). One day after 17β-estradiol pelletimplantation, 5×10⁶ cells were injected subcutaneously in a suspensioncontaining 50% phenol red-free matrigel (BD Biosciences) in Hank'sbalanced salt solution. The total injection volume containing cells insuspension was 200 μl. Mice were enrolled in the study 12 days postimplantation with average tumor volume of 197.9 mm³. After beingrandomly assigned to one of five groups (n=5/group), mice wereadministered i.v. PBS (10 ml/kg), control IgG-MCC-DM1 (15 mg/kg),unconjugated 12425 IgG (15 mg/kg), or 12425-MCC-DM1 (5, 10, or 15 mg/kg)(FIG. 15 (A)). Control IgG-MCC-DM1 was not active in this model. Asingle dose of 15 mg/kg naked IgG 12425 or 5 mg/kg 12425-MCC-DM1exhibited similar activity 29 d post dose (25 and 24% T/C,respectively). 12425-MCC-DM1 administered at both 10 and 15 mg/kgyielded tumor regression (72 and 92% regression, respectively).

The dose response antitumor activity of 12425-MCC-DM1 was evaluated inthe FGFR4 positive RH4 xenograft model. Female nude mice were implantedsubcutaneously with 10×10⁶ cells in a suspension containing 50% phenolred-free matrigel (BD Biosciences) in Hank's balanced salt solution. Thetotal injection volume containing cells in suspension was 200 μl. Micewere enrolled in the study 11 days post implantation with average tumorvolume of 200.8 mm³ (FIG. 15 (B)). After being randomly assigned to oneof five groups (n=8/group), mice were administered i.v. PBS (10 ml/kg),control IgG-MCC-DM1 (15 mg/kg), unconjugated 12425 IgG (15 mg/kg), or12425-MCC-DM1 (5, 10, or 15 mg/kg). Control IgG-MCC-DM1 was not activein this model. Similarly, a single 15 mg/kg dose of naked IgG 12425exhibited limited activity. 12425-MCC-DM1 was active as a single dose of5, 10, and 15 mg/kg (36, 24, 26% T/C respectively).

Example 19: Evaluation of Pharmacokinetics of Anti-FGFR ADCs in Mouse,Rat and Cynomolgus Monkey

The pharmacokinetics (PK) of anti-FGFR ADCs was evaluated in tumor- andnon-tumor-bearing mice at several dose levels ranging from 1-15 mg/kg,in non-tumor bearing rats at 1, 5 and 45 mg/kg and in cynomolgus monkeyat 30 mg/kg. In all studies, serum concentrations of both “total”antibody and “antibody drug conjugate (ADC)” were measured in all animalspecies using validated ELISA methods. The “total” fraction refers tothe measurement of the antibody with or without the conjugated DM1,whereas the ADC fraction refers to the measurement of DM1 conjugatedantibody only (≧1 DM1 molecule). In all studies, no appreciabledifferences were observed between clearance of the ADC and totalantibody fractions.

12425-MCC-DM1 PK was investigated in SNU16 tumor-bearing mice followinga single IV dose of 3 mg/kg. PK samples were collected at 1 h, 24 h, 72h, 168 h, and 336 h post dose and the serum concentrations of the totaland ADC fraction were super-imposable at this dose (FIG. 16 (A)).12425-MCC-DM1 half-life was about 1.5 days in the presence of the SNU16xenograft.

12425-MCC-DM1 PK was also investigated in non-tumor bearing micefollowing a single IV dose of 1, 5, 10 or 15 mg/kg. PK samples werecollected at 1 h, 6 h, 24 h, 72 h, 96 h, 168 h, 240 h. 336 h, and 540 hpost dose. The serum concentrations of the total and ADC fraction weresuper-imposable at these doses (FIG. 16 (B)) and PK properties appearedsimilar in non-tumor bearing relative to tumor bearing animals.

The PK of 12433-MCC-DM1 and 10164-MCC-DM1 in SNU16 tumor-bearing micewas also determined. The overall PK profile of these ADCs was similar tothat of 12425-MCC-DM1 and the data obtained is summarized in Table 17.

TABLE 17 Pharmacokinetic properties of 12433-, 12425- and 10164-MCC-DM1in mice at 3 mg/kg dose Cmax CL (ug/mL) (mL/h/kg) V (mL/kg) t½ (h) AbTOTAL ADC TOTAL ADC TOTAL ADC TOTAL ADC 12425-MCC-DM1 45.0 50.4 1.851.72 97.0 89.5 36.4 36.1 12433-MCC-DM1 52.4 42.7 1.61 1.95 101.0 122.943.5 43.8 10164-MCC-DM1 42.8 37.5 1.18 1.28 101.4 117.5 59.4 64.0

12425-MCC-DM1 PK was investigated in non-tumor bearing rats at two doselevels; 1 and 5 mg/kg administered IV. PK samples were collected at 0.5h, 1 h, 2 h, 6 h, 8 h and on day 1, 2, 4, 8, 11, 14, 21 and 35. Theconcentration-time profiles for the “total” and “ADC” species were nearsuperimposable at both dose levels (FIG. 16 (C)). The terminalelimination phase was slightly steeper in the 1 mg/kg dose group, andthis could be as a result of target-mediated drug disposition (TMD) atlater time-points. Similarly, clearance values for the total and ADCfractions were 0.757±0.088 mL/h/kg and 0.813±0.041 mL/h/kg, respectivelyat the 1 mg/kg dose, while slightly lower values of 0.623±0.027 mL/h/kgand 0.607±0.019 mL/h/kg, respectively were noted at the 5 mg/kg dose.12425-MCC-DM1 ADC species have a terminal half-life of around 3.9 daysat the 5 mg/kg dose level. In additional studies, rat PK was alsodetermined at 5 mg/kg and 45 mg/kg dose levels. Half-life at the 5 mg/kgdose was similar to the values noted above, while at the 45 mg/kg dosethe half-life was 163.9 h, 121.3 h (˜5 days) for the total and ADCfractions, respectively and these values were similar to observationsfor a non-FGFR2/4 cross-reactive control ADC, suggesting saturation ofany possible TMD clearance pathways.

12433-MCC-DM1 and 10164-MCC-DM1 PK was also determined in similarstudies and the PK parameters obtained are presented in Table 18. It wasfound that at the dose levels employed, similar estimates of total andADC half-life and clearance were obtained for all 3 ADCs tested.

TABLE 18 Pharmacokinetic properties of 12433-, 12425- and 10164-MCC-DM1in rats Dose AUCinf CL (mg/ t½ (h) (ug · h/mL) (mL/h/kg) kg) Ab TOTALADC TOTAL ADC TOTAL ADC 5 12433- 71.1 59.0 388.8 374.0 0.536 0.557 MCC-DM1 45 12433- 176.7 131.9 4399.3 3929.8 0.426 0.477 MCC- DM1 5 12425-77.0 64.0 12853 12874 0.389 0.388 MCC- DM1 45 12425- 163.9 121.3 103265103647 0.436 0.434 MCC- DM1 5 10164- 88.0 78.0 12677 14644 0.394 0.341MCC- DM1 45 10164- 140.1 110.3 139842 150433 0.322 0.299 MCC- DM1

12425-MCC-DM1 PK was investigated following administration of a single30 mg/kg IV dose to a group of three cynomolgus monkeys (1 male, 2female). Serum samples were collected on Day 1-0 h (prior to dosing),and at 0.25 h, 2 h, 4 h, 6 h, 24 h, 48 h, 72 h, 168 h and 240 h postdose. The maximum exposure to 12425-MCC-DM1 was observed immediatelyfollowing IV injection, i.e. at 0.25 h, which was the first samplingtime-point. The time course was characterized by a long eliminationphase, and the profiles for the total and ADC fractions weresuperimposable over the sampling time course (FIG. 16 (D)). The terminalhalf-life was around 6-7 days.

10164-MCC-DM1 PK was also investigated following administration of asingle 30 mg/kg IV dose to a group of three cynomolgus monkeys. The timecourse for this reagent was also characterized by a long eliminationphase (terminal half-life of 6-7 days) and PK parameters of both10164-MCC-DM1 and 12425-MCC-DM1 are presented in Table 19.

TABLE 19 Pharmacokinetic properties of 12425- and 0164-MCC-DM1 incynomolgus monkey following a 30 mg/kg IV dose Test Cmax AUC0-240 hAUC0-inf CL t1/2 article (μg/mL) (μg · h/mL) (μg · h/mL) (mL/h/kg) (h)12425 Total 785.6 ± 72.2 64612 ± 6477 99979 ± 12060 0.303 ± 0.038 166.5± 36.4 ADC 780.6 ± 84.2 66656 ± 7577 99113 ± 16895 0.310 ± 0.052   151 ±22.3 10164 Total 1032 ± 423 79535 ± 6506 124946 ± 11687   0.242 ± 0.0237175.1 ± 44.1 ADC  909.5 ± 224.3 81150 ± 4658 122061 ± 8615  0.247 ±0.018 158.1 ± 33  

Example 20: Assessment of the Activity of Sequence-Modified Anti-FGFRAntibodies and ADCs In Vitro and In Vivo

It is recognized that potential structural liabilities can exist in thesequences of therapeutic antibodies that can affect the heterogeneity ofthe final protein and may impact, for example, antibodymanufacturability and immunogenicity. Such liabilities can includeglycosylation sites, un-paired cysteines, potential deamidation sitesetc. To reduce the risk of such potential liabilities, mutations can beintroduced to remove one or more of these liabilities. For example,potential deamidation sites can be replaced either alone or inconjunction with other structural changes. Examples of potentialdeamidation sites include DG in heavy chain CDR2 of 10164 and 12425 (seeTable 1). These residues can be mutated to other appropriate aminoacids. By way of non-limiting example, the aspartic acid (D) in 10164HCDR2 can be mutated to glutamic acid (E) or threonine (T). In 12425,the HCDR2 glycine (G) can be mutated to another amino acid, such asalanine (A).

It should also be appreciated that where an antibody differs from itsrespective germline sequence at the amino acid level, the antibody canbe mutated back to its germline sequence. Such corrective mutations canoccur at one or more positions and be generated using standard molecularbiology techniques or by gene synthesis. By way of non-limiting example,the heavy chain sequence of 12425 heavy chain (SEQ ID NO:9, see Table 1)differs from the corresponding germline sequence by an E to a Q atposition 1, and the light chain (SEQ ID No: 17) differs from thecorresponding germline by a T for a V at position 85. Thus the aminoacids in 12425 can be modified at any or all of these sites.

The chemistry employed to generate the antibody drug conjugatesdescribed in this application relies on conjugation to lysine (K)residues in the antibody sequence. Where these lysine residues existwithin regions involved in epitope recognition, for example, in CDRregions, if significant conjugation occurs at these sites, the abilityof the antibody drug conjugate to bind to its intended target could bealtered. To mitigate this potential risk, such lysine residues can bemutated to alternative appropriate amino acids, such as arginine,asparagine or glutamine. One lysine is present in both the heavy chainCDR2 and CDR3 regions of 12425. Either one or more of these lysines canbe mutated to alternative residues, such as arginine, asparagine orglutamine.

Several of the changes described above were introduced into the 12425antibody to yield the antibody 20562 (Table 1). This antibody wascompared to 12425 for its ability to bind to FGFR2, its ability toinhibit the proliferation of FGFR2-amplified cell lines, such as SNU16and to cause an anti-tumor effect against SNU16 cells in vivo. Theaffinity of 20562 to human FGFR2 IIIb and FGFR4 was found to be similarto that of 12425 undersimilar assay conditions (20562 FGFR2 IIIbaffinity estimate: 9 nM; human FGFR4 affinity estimate: 3.3 nM).Employing the methodology described in example 6, similar to12425-MCC-DM1, 20562-MCC-DM1 was also a potent inhibitor of theproliferation of SNU16 cells in vitro (FIG. 17 (A)). To assess thepotency of 20437-MCC-DM1 in vivo, female nude mice were implantedsubcutaneously with 10×10⁶ cells in a suspension containing 50% phenolred-free matrigel (BD Biosciences) in Hank's balanced salt solution. Thetotal injection volume containing cells in suspension was 200 al. Micewere enrolled in the study seven days post implantation with an averagetumor volume of 217.1 mm³. After being randomly assigned to one of fivegroups (n=6/group), mice received PBS (10 ml/kg) or a 3 mg/kg i.v. doseof 12425-MCC-DM1 or 20562-MCC-DM1. Tumors were calipered twice per week(FIG. 17(B)). Both 12425-MCC-DM1 and its variant 20562-MCC-DM1 hadsimilar activity in vivo against the FGFR2 amplified SNU16 xenograft.These data suggest that it is possible to remove the DG site of theparent antibody without impacting in vivo activity as an ADC.

Example 21: Generation and Characterization of Affinity Matured FGFRADCs

To evaluate the effect of affinity on the biological activity ofselected anti-FGFR antibodies, affinity optimization was performed onthe 12433, 10164 and 12425 clones. In these studies, the L-CDR3 andH-CDR2 regions were optimized in parallel by cassette mutagenesis usingtrinucleotide directed mutagenesis (Virnekas et al., 1994 Nucleic AcidsResearch 22: 5600-5607), while the framework regions were kept constant.Prior to cloning for affinity maturation, parental Fab fragments weretransferred from the corresponding expression vector pM®x11 into theCysDisplay™ vector pMORPH®30 via XbaI/EcoRI.

For optimizing L-CDR3 of parental Fab fragments, the L-CDR3 of thebinder was removed and replaced by a repertoire of diversified L-CDR3s.For each parental Fab, a second library set with diversified H-CDR2 hasbeen generated. For each maturation library (LCDR3 and HCDR2),antibody-displaying phages were prepared and phage titers weredetermined by spot-titration. Amplification of the library was performedas described elsewhere (Rauchenberger et al., 2003 J Biol Chem 278:38194-38205). For quality control, single clones were randomly pickedand sequenced.

For the selection of affinity improved binders phage derived frommaturation libraries were subjected to three rounds of solution panningsusing biotinylated human FGFR2. Stringency was increased by lowering theantigen concentration in each panning round (Low et al., 1996 J Mol Biol260(3): 359-368). In addition to antigen reduction, off-rate selection(Hawkins et al., 1992 J Mol Biol 226: 889-896) was performed. This wascombined with prolonged washing steps at RT. The general panningprocedure was performed as described above (solution panning).

For selected subcodes, the 2^(nd) round panning output of a conductedH-CDR2 affinity maturation panning was diversified additionally in theL-CDR3. Library generation was exactly done as described above. For theselection of improved binders, the new generated libraries weresubjected into two additional rounds of solution panning.

The sequence unique clones that were identified were expressed as IgGsand affinity to human FGFR2 was determined using either SET or Biacoremethodologies similar to those described in examples 1 and 2. ore than80 clones were evaluated from the 3 parental antibodies and affinityimprovements of 3-6 fold were obtained for clones derived from the 12433antibody, 27-fold for 10164 derived clones and 68-fold for 12425 derivedantibodies. The highest affinity clone derived from 10164 was designated20809 and it's affinity for human FGFR2 was determined to be 450 pM.Interestingly, this antibody also bound to human FGFR4 with an affinityestimate of 7.4 nM. The highest affinity antibody derived from 12425 wasdesignated 20811 (Table 1) and it's affinity for human FGFR2 wasdetermined to be 110 pM. The affinity of this antibody to FGFR4 wasdetermined to be 75 pM.

In additional studies, both 20809 and 20811 were directly conjugated toSMCC-DM1 to yield 20809-MCC-DM1 and 20811-MCC-DM1. The ability of theseADCs to inhibit the growth of SNU16 cells is shown in FIG. 18 (A). Theimpact of affinity on in vivo antitumor activity was assessed in theFGFR2 amplified SNU16 xenograft model. Female nude mice were implantedsubcutaneously with 10×10⁶ cells in a suspension containing 50% phenolred-free matrigel (BD Biosciences) in Hank's balanced salt solution. Thetotal injection volume containing cells in suspension was 200 μl. Micewere enrolled in the study seven days post implantation with an averagetumor volume of 217.6 mm³. After being randomly assigned to one of fivegroups (n=6/group), mice received PBS (10 ml/kg) or a 3 mg/kg i.v. doseof 12425-MCC-DM1, 20811-MCC-DM1, 10164-MCC-DM1, or 20809-MCC-DM1. Tumorswere calipered twice per week (FIG. 18 (B)). 10164-MCC-DM1 and itsaffinity matured variant 20809-MCC-DM1 demonstrated similar activityagainst the SNU16 xenograft. 12425-MCC-DM1 demonstrated superiority overits affinity matured variant 20811-MCC-DM1 with regards to antitumoractivity. These data suggest that affinity maturation did not yieldimproved activity in vivo as MCC-DM1 ADCs.

Example 22: Epitope Mapping of FGFR2 and its Antibody Complex byDeuterium Exchange Mass Spectrometry (HDx-MS)

Deuterium exchange mass spectrometry (HDx-MS) measures the deuteriumuptake on the amide backbone of a protein. These measurements aresensitive to the amide's solvent accessibility and to changes in thehydrogen bonding network of the backbone amides. HDx-MS is often used tocompare proteins in two different states, such as apo and ligand-bound,and coupled with rapid digestion with pepsin. In such experiments onecan locate regions, typically of 10 to 15 amino acids, that showdifferential deuterium uptake between two different states. Regions thatare protected are either directly involved in ligand binding orallosterically affected by binding of the ligand.

In these experiments, the deuterium uptake of E. Coli. derived FGFR2D2-D3 protein (SEQ ID NO: 135, see below) was measured in the absenceand presence of three therapeutic antibodies: 12425, 10164, and 12433.Regions in FGFR2 that show a decrease in deuterium uptake upon bindingof the antibody are likely to be involved in the epitope; however, dueto the nature of the measurement it is also possible to detect changesremote from the direct binding site (allosteric effects). Usually, theregions that have the greatest amount of protection are involved indirect binding although this may not always be the case. In order todelineate direct binding events from allosteric effects orthogonalmeasurements (e. g. X-ray crystallography, alanine mutagenesis) arerequired.

TABLE 17 FGFR2 D2-D3 Construct SEQ ID NO: 135 LENGTH: 237 amino acidsTYPE: Protein ORGANISM: HumanMAEDFVSENSNNKRAPYWTNTEKMEKRLHAVPAANTVKFRCPAGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESVVPSDKGNYTCVVENEYGSINHTYHLDVVERSPHRPILQAGLPANASTVVGGDVEFVCKVYSDAQPHIQWIKHVEKNGSKYGPDGLPYLKVLKHSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQSAWLTVLPKQQAPGREKEHHHHHH

The epitope mapping experiments described in this example were performedon a Waters HDx-MS platform, which includes a LEAP autosampler,nanoACQUITY UPLC System, and Synapt G2 mass spectrometer. The studieswere automated by a LEAP autosampler operated by the LeapShell software,which performed initiation of the deuterium exchange reaction, reactiontime control, quench reaction, injection onto the UPLC system anddigestion time control. The Leap autosampler was equipped with twotemperature controlled stacks maintained at 25° C. for HDx reaction andmaintained at 2° C. for storage of protein and quench solution,respectively. Triplicate control experiments were carried out on antigenat a deuterium exchange time of 25 minutes. HDX was quenched withquenching buffer (6 M urea and 1 M TCEP pH=2.5). After quenching, theantigen was injected into the UPLC system where it is subjected toon-line pepsin digestion at 12° C. followed by a rapid 8 min 2 to 35%acetonitrile gradient over a Waters BEH C18 1×100 mm column (maintainedat 1° C.) at a flow rate of 40 μL/min. Triplicate experiments werecarried out on antigen-mAb complex just as the control experimentsexcept in these experiments the antigen in incubated for 30 min with theantibody at 25° C. prior to deuterium exchange.

The results of these measurements are summarized in FIG. 19 (A) and FIG.19 (B). In FIG. 19 (A) the average deuterium uptake for FGFR2 peptidesin the absence (control) and presence of mAbs is indicated. In thisFigure it is useful to examine two differences: differences betweencontrol and mAbs and differences among mAb groups. In FIG. 19 (B), thedifference in the deuterium uptake between the mAb and control samplesis divided by the standard error in the measurement. Differences areconsidered significant if they are greater than 0.5 Da in absolute scale(FIG. 19 (A)) and if the ratio of the difference divided by the standarderror is less than or equal to −3.0 (FIG. 19 (B)). From this analysis wecan rank order regions of the FGFR2 that are protected upon mAb bindinginto categories of high, medium, and low amounts of protection. FGFR2regions of high protection are potentially involved in the formation ofthe epitope with a specific antibody although one cannot rule out othercontributions for lesser protected regions.

Upon binding of 10164 to FGFR2 we observe high amounts of protection inthe following two regions of FGFR2: 174-189 and 198-216. These regionsare located in the D2 domain specifically consisting of βB to N-terminalside of βC and βC′ loop to the βE (FIGS. 19 (C) and (D)). There is oneregion of low protection found in the regions 222-231. However, thisregion is protected upon binding of all studied antibodies; thisobservation suggests that this region undergoes a stabilization of itslocal hydrogen bonding network that may be non-specific and allostericin nature. Binding of the antibody 12425 to FGFR2 also cause a highamount of protection in the regions 174-189 and 198-216. In addition,with 12425 family a high amount of protection in the region 160-173(FIGS. 19 (A) and (B)) was also observed. These regions comprises aregion of FGFR2 that is known to interact with FGF2 and FGF1 frompublished crystallography data (Plotnikov et al., M. Cell 2000, 101,413-424; Beenken et. al., M. J. Biol. Chem. 2012, 287, 3067-3078).Protection in this region is unique to the 12425 family of antibodies.Of the antibodies studied, 12433 is unique in that it specificallytargets the IIIb isoform of FGFR2 through interactions with the D3domain of FGFR2. For 12433 high amounts of protection (˜2 Da) in in thepeptide 338-354, and insignificant amounts of protection in a shorterN-terminal fragment from 338-345 were observed. This observationsuggests that the portion of the 338-354 peptide that is protected upon12433 binding is the region 346-354 (FIGS. 19 (A) and (B)). The region346 to 354 contains four residues that are specific to IIIb isoformrelative to the IIIc isoform; these residues include: Q348, A349, N350,and Q351. In additional studies, affinity matured versions of 10164 and12425 (see Example 23 below) were evaluated and were found to be similarto their respective parental antibodies in terms of the protectedregions observed.

Additionally, using similar methods deuterium exchange studies examiningthe protection of FGFR4 in the presence of FGFR2/4 cross-reactiveantibody 12425 were performed. Preliminary analysis using an FGFR4 D1-D3construct (SEQ ID NO: 124, see below) suggested that binding of MOR12425does not cause any protection in the D3 region. Protection appears to belimited to regions within the D1 and D2 domains.

TABLE 18 FGFR4 D1-D3 Construct SEQ ID NO: 124 LENGTH: 371 amino acidsTYPE: Protein ORGANISM: HumanLEASEEVELEPCLAPSLEQQEQELTVALGQPVRLCCGRAERGGHWYKEGSRLAPAGRVRGWRGRLEIASFLPEDAGRYLCLARGSMIVLQNLTLITGDSLTSSNDDEDPKSHRDPSNRHSYPQQAPYWTHPQRMEKKLHAVPAGNTVKFRCPAAGNPTPTIRWLKDGQAFHGENRIGGIRLRHQHWSLVMESVVPSDRGTYTCLVENAVGSIRYNYLLDVLERSPHRPILQAGLPANTTAVVGSDVELLCKVYSDAQPHIQWLKHIVINGSSFGADGFPYVQVLKTADINSSEVEVLYLRNVSAEDAGEYTCLAGNSIGLSYQSAWLTVLPEEDPTWTAAAPEARYTDKLEFRHDSGLNDIFEAQKIEWHE

Example 23: X-Ray Crystallographic Structure Determination of the HumanFGFR2/12425 Fab Complex

The crystal structure of a human FGFR2 ECD fragment (domain 2, or D2,SEQ ID NO: 135, Table 19) bound to Fab fragment of 12425 (Table 19) aredetermined. As detailed below, FGFR2 D2 is expressed, refolded, purifiedand mixed with 12425 Fab to form a complex. Protein crystallography wasthen employed to generate atomic resolution data for FGFR2 D2 bound to12425 Fab to define the epitope.

Protein Production of FGFR D2 and 12425 Fab for Crystallography

The sequences of FGFR2 D2 and 12425 Fab produced for crystallography areshown in Table 19. Construct of FGFR2 D2 comprises residues 146 to 249(underlined) of human FGFR2 (UniProt identifier P21802-3, SEQ ID NO:137), along with N- and C-terminal residues from recombinant expressionvector (shown in lower case letters, SEQ ID NO: 137). For 12425 Fab, thesequences of heavy and light chains are shown (SEQ ID NO: 139 and 140,respectively).

TABLE 19 Proteins used for crystal structure determinationAmino acid sequence SEQ Construct in one letter code ID NO Human FGFR2MVSWGRFICLVVVTMATLSLARPSFSLVE 137 (P21802-3)DTTLEPEEPPTKYQISQPEVYVAAPGESL EVRCLLKDAAVISWTKDGVHLGPNNRTVLIGEYLQIKGATPRDSGLYACTASRTVDSE TWYFMVNVTDAISSGDDEDDTDGAEDFVSENSNNKRAPYWTNTEKMEKRLHAVPAANT VKFRCPAGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESVVPSDKGNYTCV VENEYGSINHTYHLDVVERSPHRPILQAGLPANASTVVGGDVEFVCKVYSDAQPHIQW IKHVEKNGSKYGPDGLPYLKVLKHSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQ ANQSAWLTVLPKQQAPGREKEITASPDYLEIAIYCIGVFLIACMVVTVILCRMKNTTK KPDFSSQPAVHKLTKRIPLRRQVTVSAESSSSMNSNTPLVRITTRLSSTADTPMLAGV SEYELPEDPKWEFPRDKLTLGKPLGEGCFGQVVMAEAVGIDKDKPKEAVTVAVKMLKD DATEKDLSDLVSEMEMMKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLRAR RPPGMEYSYDINRVPEEQMTFKDLVSCTYQLARGMEYLASQKCIHRDLAARNVLVTEN NVMKIADFGLARDINNIDYYKKTTNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLMWE IFTLGGSPYPGIPVEELFKLLKEGHRMDKPANCTNELYMMMRDCWHAVPSQRPTFKQL VEDLDRILTLTTNEEYLDLSQPLEQYSPSYPDTRSSCSSGDDSVFSPDPMPYEPCLPQ YPHINGSVKT FGFR2 D2mNSNNKRAPYWTNTEKMEKRLHAVPAANT 138 VKFRCPAGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESVVPSDKGNYTCV VENEYGSINHTYHLDVVlvprgslehhhh hh12425 Fab heavy QVQLLESGGGLVQPGGSLRLSCAASGFTF 139 chainSDYAMSWVRQAPGKGLEWVSVIEGDGSYT HYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKTYSSAFDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH 12425 Fab light DIQMTQSPSSLSASVGDRVTITCRASQDI140 chain SSDLNWYQQKPGKAPKLLIYDASNLQSGV PSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYSPSHTFGQGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC

FGFR2 D2 was expressed in E. coli BL21 (DE3) (Novagen®, EMD Millipore).Following overnight induction with IPTG at 18° C., cells were harvestedand lysed. The inclusion body was extracted by incubation with 20 mMsodium phosphate pH 7.5, 300 mM NaCl and 8M urea, and loaded onto aNi-NTA column pre-equilibrated in the same buffer. The column was washedwith 20 mM sodium phosphate pH 7.5, 300 mM NaCl, 50 mM ammonium sulfateand 20 mM imidazole, followed by elution with 20 mM sodium phosphate pH7.5, 300 mM NaCl, 50 mM ammonium sulfate and 300 mM imidazole. Theeluted protein was then diluted 12-fold with 20 mM sodium phosphate pH6.5 (buffer A), and loaded onto HiTrap S HP column (GE Healthcare),pre-equilibrated in buffer A plus 2% of 20 mM sodium phosphate pH 6.5,1.5M NaCl, 50 mM ammonium sulfate (buffer B). The S column was eluted bya gradient of 2%-100% buffer B. The major peak containing FGFR D2 wascollected, concentrated, and loaded onto HiLoad 16/60 Superdex 75 (GEHealthcare) equilibrated in 20 mM Hepes pH 7.5, 150 mM NaCl. Peakfractions were analyzed by SDS-PAGE and LCMS and then pooled to form acomplex with 12425 Fab.

12425 Fab is produced by cleavage of full-length 12425 IgG withimmobilized papain (Pierce). 12425 IgG at 20 mg/ml in 20 mM sodiumphosphate pH 7.0 and 10 mM EDTA was mixed with immobilized papain at aweight ratio of 80:1. The mixture was rotated in a 15 ml tube at 37° C.overnight. The next day the immobilized papain was removed by gravityflow column; the flow through, which contains both Fab and Fc segments,was collected and loaded onto HiTrap MabSelect SURE column (GEHealthcare) to remove the Fc segment. The flow through from this step,which contains only the Fab fragment, was concentrated and loaded ontoHiLoad 16/60 Superdex 75 (GE Healthcare) equilibrated in 20 mM Hepes pH7.5, 150 mM NaCl. Peak fractions were analyzed by SDS-PAGE and LCMS, andthen pooled to form a complex with FGFR D2.

Crystallization and Structure Determination of FGFR2 D2/12425 FabComplex

A complex of FGFR2 D2 with 12425 Fab was prepared by mixing the purifiedFGFR2 D2 and 12425 Fab at a 2:1 molar ratio (concentration measured viaLCUV), incubating on ice for 30 min, and purifying the complex on aHiLoad 16/60 Superdex 75 (GE Healthcare) equilibrated in 20 mM Hepes pH7.5, 150 mM NaCl. Peak fractions were analyzed by SDS-PAGE and LCMS.Fractions containing FGFR2 D2/12425 Fab complex were concentrated toabout 30 mg/ml. Trypsin (dissolved in 1 mM HCl and 2 mM CaCl₂ at 1mg/ml) was added into the complex at a volume ratio of 1:100 tofacilitate crystallization (Wernimont et al., (2009) Plos One 4:e5095).The FGFR2 D2/12425 Fab/trypsin mixture was immediately centrifuged andscreened for crystallization.

Crystals were grown by sitting drop vapor diffusion. In detail, 0.1 μlof protein was mixed with 0.1 μl of reservoir solution which contains0.1 M tri-sodium citrate dihydrate pH 5.0, 20% (w/v) PEG6000; and thedrop was equilibrated against 45 μl of the same reservoir solution at20° C.

Before data collection, the FGFR2 D2/12425 Fab crystals was transferredto a reservoir solution containing an additional 22.5% glycerol andflash cooled in liquid nitrogen. Diffraction data were collected atbeamline 17-ID at the Advanced Photon Source (Argonne NationalLaboratory, USA). Data were processed and scaled at 2.8 Å using HKL2000(HKL Research) in space group C2 with cell dimensions a=228.79 Å,b=96.94 Å, c=200.66 Å, alpha=90°, beta=106.110, gamma=90°. The FGFR2D2/12425 Fab complex structure was solved by molecular replacement usingPhaser (McCoy et al., (2007) J. Appl. Cryst. 40:658-674) with FGFR2 D2structure (PDB ID: 3DAR) and an in-house Fab structure as search models.The final model was built in COOT (Emsley & Cowtan (2004) Acta Cryst.60:2126-2132) and refined with Buster (Global Phasing, LTD) to R_(work)and R_(free) values of 22.6% and 24.9%, respectively, with an rmsd of0.009 Å and 1.1° for bond lengths and bond angles, respectively.Residues of FGFR2 D2 that contain atoms within 5 Å of any atom in 12425Fab are identified by PyMOL (Schrödinger, LLC) and listed in Tables 20and 21.

FGFR2 Epitope for 12425

The crystal structure of the FGFR2 D2/12425 Fab complex was used toidentify the FGFR2 epitope for 12425. There are six copies of 12425Fab-FGFR2 D2 complex in the asymmetric unit of the crystal (anasymmetric unit contains all the structural information neede toreproduce the whole crystal). All six copies share almost identicalresidues in contact with 12425 Fab except for small variations due tocrystal packing. Only those 12425-contacting FGFR2 residues that areshared by all six copies were used to define epitope.

The interaction surface on FGFR2 D2 by 12425 Fab is formed by severaldiscontinuous (i.e., noncontiguous) sequences: namely residues 173through 176, residue 178, residues 208 through 210, residues 212, 213,217 and 219, as detailed in Table 20 and 21 These residues form thethree-dimensional surface that is recognized by 12425 Fab (FIG. 20 (A)).This epitope defined by crystallography is in good agreement with thatdefined by deuterium exchange mass spectrometry (HDx-MS), whichcomprises residues 160-173, 174-189 and 198-216.

TABLE 20 Interactions between human FGFR2 D2 and 12425 Fab heavy chain(H). FGFR2 residues are numbered based upon P21802-3 (SEQ ID NO: 137).Fab heavy chain residues are numbered ased upon their linear amino acidsequence (SEQ ID NO: 139). FGFR2 residues shown have at least one atomwithin 5 Å of an atom in the 12425 Fab, to account for potential watermediated interactions. Human FGFR2 12425 Fab Amino acid Number Aminoacid Number Chain LYS 208 HIS 59 H VAL 209 TYR 57 H ARG 210 ALA 33 H VAL50 H TYR 57 H GLU 52 H GLU 99 H SER 104 H GLN 212 GLU 52 H ASP 54 H HIS213 GLU 52 H SER 104 H

TABLE 21 Interactions between human FGFR D2 and 12425 Fab light chain(L). FGFR2 residues are numbered based upon P21802-3 (SEQ ID NO: 137).Fab light chain residues are numbered based upon their linear amino acidsequence (SEQ ID NO: 140). FGFR2 residues shown have at least one atomwithin 5 Å of an atom in the 12425 Fab, to account for potential watermediated interactions. Human FGFR2 12425 Fab Residue Number ResidueNumber Chain ASN 173 GLN 27 L THR 174 GLN 27 L TYR 92 L VAL 175 TYR 92 LLYS 176 ASP 32 L HIS 91 L TYR 92 L ASP 32 L ARG 178 ASP 32 L ARG 210 HIS96 L ILE 217 TYR 92 L PRO 94 L GLU 219 SER 93 L PRO 94 L

sites on FGFR2, namely Asn241 and Asn288 (Duchesne et al., (2006) J.Biol. Chem 281:27178-27189) are on the opposite surface of the proteinaway from the 12425 epitope (FIG. 20 (A)), indicating that 12425 bindingto FGFR2 is independent of glycosylation. This is consistent with thefinding that 12425 antibody binds E. coli-produced (no glycosylation)and mammalian cell-produced (glycosylated) FGFR2 D2-D3 with similaraffinity (data not shown).

Lys176 and Arg210 of FGFR2 D2 are the two epitope residues making mostcontacts with 12425 Fab light and heavy chains, respectively.Interestingly, Arg210 has been demonstrated to bind heparin (Pellegriniet al., (2000) Nature 407:1029-1034), which can enhance FGFR2 binding toFGFs and subsequent dimerization and signaling. Meanwhile, Lys176 isalso postulated to be in the heparin binding pocket (Pellegrini et al.,(2000) Nature 407:1029-1034).

Two models have been reported for how FGFR-FGF-heparin complex dimerizeson the cell surface to activate downstream signaling, namely the 2:2:1(FGFR:FGF:heparin) model (Pellegrini et al., (2000) Nature407:1029-1034) and the 2:2:2 model (Schlessinger et al., (2000) Mol.Cell 6:743-750). Based on the crystal structure of FGFR2 D2/12425 Fabcomplex, the binding of 12425 antibody to FGFR2 clashes with and thuscan block both dimerization models (FIGS. 20 (B) and (C)).

Based on the sequence alignment of human FGFR2 and FGFR4 (FIG. 20 (D)),9 out of the 12 residues on FGFR2 D2 contacting 12425 Fab in the crystalstructure are completely conserved in FGFR4; the other 3 are partiallyconserved (of similar type of sidechains (e.g. basic, hydrophobic)).This provides a structural basis for the ability of 12425 to be able tobind to both FGFR2 and FGFR4.

Example 24. Formulation

The clinical service form (CSF) of the ADC is a lyophilisate in vialcontaining 50 mg 12425-MCC-DM1, 16.2 mg sodium succinate, 410.8 mgsucrose and 1 mg polysorbate 20 (without considering the overfill of 10%to allow for withdrawal of the declared content). After reconstitutionof the lyophilizate with 5 mL water for injection, a solution containing10 mg/mL 12425-MCC-DM1, 20 mM sodium succinate, 240 mM Sucrose and 0.02%polysorbate 20 at a pH of 5.0 is obtained.

For subsequent intravenous administration, the obtained solution willusually be further diluted into a carrier solution to the ready-to-useADC solution for infusion.

For the CSF, an ADC concentration of 10 mg/mL was chosen based onpreliminary stability testing. A sucrose concentration of 240 mM wasselected in order to create an isotonic formulation, to maintain anamorphous lyophilizate cake structure and to afford proteinstabilization.

Important stability-indicating analytical methods to select the moststable formulation encompassed, amongst others, size-exclusionchromatography to determine aggregation levels, subvisible particulatematter testing, free Toxin determination and potency testing.

The pre-screening study showed that polysorbate 20 at a concentration of0.02% provides sufficient stabilization against mechanical stress. Theliquid and lyophilized stability studies at real-time and acceleratedstability conditions (25° C. and 40° C.) demonstrated that a succinatepH 5.0 formulation provides the overall best storage stability. Mostnotably in this formulation the best balance of all tested formulationsbetween aggregation and release of the free Toxin could be met. Afterthree months at 40° C. no noteworthy increase in degradation productscould be determined.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

1. An antibody drug conjugate of the formulaAb-(L-(D)_(m))_(n) or a pharmaceutically acceptable salt thereof;wherein Ab is an antibody or antigen binding fragment thereof thatspecifically binds to both human FGFR2 and FGFR4; L is a linker; D is adrug moiety; m is an integer from 1 to 8; and n is an integer from 1 to10.
 2. The antibody drug conjugate of claim 1, wherein said m is
 1. 3.The antibody drug conjugate of claim 1, wherein said n is 3 or
 4. 4. Theantibody drug conjugate of claim 1, wherein said antibody or antigenbinding fragment specifically binds to all isoforms of human FGFR2. 5.The antibody drug conjugate of claim 1, wherein said antibody or antigenbinding fragment specifically binds to an epitope on human FGFR2comprising amino acid residues 176 (Lys) and 210 (Arg) of SEQ ID NO:137.6. The antibody drug conjugate of claim 1, wherein said antibody orantigen binding fragment recognizes amino acid residues 173 (Asn), 174(Thr), 175 (Val), 176 (Lys), 178 (Arg), 208 (Lys), 209 (Val), 210 (Arg),212 (Gln), 213 (His), 217 (Ile), and 219 (Glu) of SEQ ID NO:137.
 7. Theantibody drug conjugate of claim 1, wherein said antibody or antigenbinding fragment specifically binds to an epitope of human FGFR2comprising SEQ ID NO:136 or SEQ ID NO:141.
 8. The antibody drugconjugate of claim 1, wherein said antibody or antigen binding fragmentspecifically binds to an epitope of human FGFR2 consisting of SEQ IDNO:136 or SEQ ID NO:141.
 9. The antibody drug conjugate of claim 1,wherein said antibody or antigen binding fragment specifically binds toD1 and D2 domains of human FGFR4.
 10. (canceled)
 11. (canceled) 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. (canceled)17. (canceled)
 18. The antibody drug conjugate of claim 1, wherein saidantibody is a human antibody.
 19. The antibody drug conjugate of claim1, wherein said antibody is a monoclonal antibody.
 20. The antibody drugconjugate of claim 1, wherein said linker is selected from the groupconsisting of a cleavable linker, a non-cleavable linker, a hydrophiliclinker, a procharged linker and a dicarboxylic acid based linker. 21.The antibody drug conjugate of claim 20, wherein the linker is derivedfrom a cross-linking reagent selected from the group consisting ofN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl4-(2-pyridyldithio)butanoate (SPDB),N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB),N-succinimidyl iodoacetate (SIA),N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS,N-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC),N-sulfosuccinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate(sulfo-SMCC) or 2,5-dioxopyrrolidin-1-yl17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate(CX1-1).
 22. The antibody drug conjugate of claim 21, wherein saidlinker is derived from the cross-linking reagent N-succinimidyl4-(maleimidomethyl)cyclohexanecarboxylate (SMCC).
 23. The antibody drugconjugate of claim 1, wherein said drug moiety is selected from a groupconsisting of a V-ATPase inhibitor, a pro-apoptotic agent, a Bcl2inhibitor, an MCL1 inhibitor, a HSP90 inhibitor, an IAP inhibitor, anmTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, anauristatin, a dolastatin, a maytansinoid, a MetAP (methionineaminopeptidase), an inhibitor of nuclear export of proteins CRM1, aDPPIV inhibitor, proteasome inhibitors, inhibitors of phosphoryltransfer reactions in mitochondria, a protein synthesis inhibitor, akinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesininhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylatingagent, a DNA intercalator, a DNA minor groove binder and a DHFRinhibitor.
 24. The antibody drug conjugate of claim 23, wherein thecytotoxic agent is a maytansinoid.
 25. The antibody drug conjugate ofclaim 24, wherein the maytansinoid isN(2′)-deacetyl-N(2′)-(3-mercapto-1-oxopropyl)-maytansine (DM1) orN(2′)-deacetyl-N2-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).26. The antibody drug conjugate of claim 1 having the following formula:

wherein Ab is an antibody or antigen binding fragment thereof comprisinga heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2,a heavy chain CDR3 of SEQ ID NO: 3, and a light chain CDR1 of SEQ ID NO:11, a light chain CDR2 of SEQ ID NO: 12, a light chain CDR3 of SEQ IDNO: 13, wherein the CDR is defined in accordance with the Kabatdefinition; and n is 1 to 10; or a pharmaceutically acceptable saltthereof.
 27. A pharmaceutical composition comprising the antibody drugconjugate of claim 1 and a pharmaceutically acceptable carrier.
 28. Thepharmaceutical composition of claim 27 wherein said composition isprepared as a lyophilisate.
 29. The pharmaceutical composition of claim28, wherein said lyophilisate comprises said antibody drug conjugate,sodium succinate, and polysorbate
 20. 30. A method of treating an FGFR2positive or FGFR4 positive cancer in a patient in need thereof,comprising administering to said patient the antibody drug conjugate ofclaim
 1. 31. The method of claim 30, wherein said cancer is selectedfrom the group consisting of gastric cancer, breast cancer, alveolarrhabdomyosarcoma, liver cancer, adrenal cancer, lung cancer, coloncancer and endometrial cancer.
 32. The method of claim 30 furthercomprising administering to said patient a tyrosine kinase inhibitor, anIAP inhibitor, a Bcl2 inhibitor, an MCL1 inhibitor, or another FGFR2inhibitor.
 33. The method of claim 32, wherein said another FGFR2inhibitor is3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methyl-urea.34. (canceled)
 35. (canceled)
 36. An antibody or antigen bindingfragment thereof that specifically binds to FGFR2 and FGFR4.
 37. Theantibody or antigen binding fragment of claim 36, wherein said antibodyor antigen binding fragment specifically binds to an epitope on humanFGFR2 comprising amino acid residues 176 (Lys) and 210 (Arg) of SEQ IDNO:137.
 38. (canceled)
 39. (canceled)
 40. The antibody or antigenbinding fragment of claim 37, wherein said antibody or antigen bindingfragment specifically binds to D1 and D2 domains of human FGFR4. 41.(canceled)
 42. The antibody or antigen binding fragment of claim 36comprising a heavy chain variable region that comprises: (a) a VH CDR1of SEQ ID NO: 1, (b) a VH CDR2 of SEQ ID NO: 2, and (c) a VH CDR3 of SEQID NO: 3, wherein the CDR is defined in accordance with the Kabatdefinition.
 43. The antibody or antigen binding fragment of claim 42further comprising a light chain variable region that comprises: (a) aVL CDR1 of SEQ ID NO: 11, (b) a VL CDR2 of SEQ ID NO: 12, and a VL CDR3of SEQ ID NO: 13, wherein the CDR is defined in accordance with theKabat definition.
 44. (canceled)
 45. (canceled)
 46. (canceled) 47.(canceled)
 48. The antibody or antigen binding fragment of claim 36,wherein said antibody is a human antibody.
 49. The antibody or antigenbinding fragment of claim 36, wherein said antibody is a monoclonalantibody.
 50. The antibody or antigen binding fragment of claim 36,wherein said antibody or antigen binding fragment is a single chainantibody (scFv).
 51. A nucleic acid that encodes the antibody or antigenbinding fragment of claim
 36. 52. A vector comprising the nucleic acidof claim
 51. 53. A host cell comprising the vector according to claim52.
 54. A process for producing an antibody or antigen binding fragmentcomprising cultivating the host cell of claim 53 and recovering theantibody from the culture.
 55. A process for producing an anti-FGFR2 andFGFR4 antibody drug conjugate comprising: (a) chemically linking SMCC toa drug moiety DM-1; (b) conjugating said linker-drug to the antibodyrecovered from the cell culture of claim 54; and (c) purifying theantibody drug conjugate.
 56. The antibody drug conjugate made accordingto claim 55 having an average DAR, measured with a UV spectrophotometer,about 3.5.
 57. A diagnostic reagent comprising the antibody or antigenbinding fragment thereof of claim 36, which is labeled.
 58. Thediagnostic reagent of claim 57, wherein the label is selected from thegroup consisting of a radiolabel, a fluorophore, a chromophore, animaging agent, and a metal ion.
 59. The antibody drug conjugate of claim1, wherein said antibody or antigen binding fragment thereof comprises aheavy chain variable region that comprises: (a) a VH CDR1 of SEQ ID NO:81, (b) a VH CDR2 of SEQ ID NO: 82, and (c) a VH CDR3 of SEQ ID NO: 83,wherein the CDR is defined in accordance with the Kabat definition. 60.The antibody drug conjugate of claim 59, wherein said antibody orantigen binding fragment thereof further comprises a light chainvariable region that comprises: (a) a VL CDR1 of SEQ ID NO: 91, (b) a VLCDR2 of SEQ ID NO: 92, and (c) a VL CDR3 of SEQ ID NO: 93, wherein theCDR is defined in accordance with the Kabat definition.
 61. The antibodydrug conjugate of claim 60, wherein said antibody or antigen bindingfragment thereof comprises a VH region of SEQ ID NO: 87 and a VL regionof SEQ ID NO:
 97. 62. The antibody drug conjugate of claim 61, whereinsaid antibody consists of a heavy chain of SEQ ID NO: 89 and a lightchain of SEQ ID NO:
 99. 63. The antibody of claim 36, wherein saidantibody or antigen binding fragment thereof comprises a heavy chainvariable region that comprises: (a) a VH CDR1 of SEQ ID NO: 81, (b) a VHCDR2 of SEQ ID NO: 82, and (c) a VH CDR3 of SEQ ID NO: 83, wherein theCDR is defined in accordance with the Kabat definition.
 64. The antibodyof claim 63, wherein said antibody or antigen binding fragment thereoffurther comprises a light chain variable region that comprises: (a) a VLCDR1 of SEQ ID NO: 91, (b) a VL CDR2 of SEQ ID NO: 92, and (c) a VL CDR3of SEQ ID NO: 93, wherein the CDR is defined in accordance with theKabat definition.
 65. The antibody of claim 64, wherein said antibody orantigen binding fragment thereof comprises a VH region of SEQ ID NO: 87and a VL region of SEQ ID NO:
 97. 66. The antibody of claim 65, whereinsaid antibody consists of a heavy chain of SEQ ID NO: 89 and a lightchain of SEQ ID NO: 99.